EP1934261B1 - Human monoclonal antibodies to cd70 - Google Patents

Human monoclonal antibodies to cd70 Download PDF

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EP1934261B1
EP1934261B1 EP06815627.2A EP06815627A EP1934261B1 EP 1934261 B1 EP1934261 B1 EP 1934261B1 EP 06815627 A EP06815627 A EP 06815627A EP 1934261 B1 EP1934261 B1 EP 1934261B1
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antibody
human
cell
antibodies
cells
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French (fr)
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EP1934261A2 (en
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Jonathan Alexander Terrett
Li-Sheng Lu
David John King
Josephine M. Cardarelli
Chin Pan
Haichun Huang
Marco A. Coccia
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ER Squibb and Sons LLC
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Medarex LLC
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2875—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF/TNF superfamily, e.g. CD70, CD95L, CD153, CD154
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/40—Immunoglobulins specific features characterized by post-translational modification
    • C07K2317/41—Glycosylation, sialylation, or fucosylation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/77—Internalization into the cell
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the cytokine receptor CD27 is a member of the tumor necrosis factor receptor (TFNR) superfamily, which play a role in cell growth and differentiation, as well as apoptosis or programmed cell death.
  • the ligand for CD27 is CD70, which belongs to the tumor necrosis factor family of ligands.
  • CD70 is a 193 amino acid polypeptide having a 20 amino acid hydrophilic N-terminal domain and a C-terminal domain containing 2 potential N-linked glycosylation sites ( Goodwin, R.G. et al. (1993) Cell 73:447-56 ; Bowman et al. (1994) Immunol 152:1756-61 ). Based on these features, CD70 was determined to be a type II transmembrane protein having an extracellular C-terminal portion.
  • CD70 is transiently found on activated, but not resting T and B lymphocytes and dendritic cells ( Hintzen et al. (1994) J Immunol. 152:1762-1773 ; Oshima et al. (1998) Int. Immunol. 10:517-26 ; Tesselaar et al. (2003) J Immunol. 170:33-40 ).
  • CD70 expression has been reported in different types of cancers including renal cell carcinomas, metastatic breast cancers, brain tumors, leukemias, lymphomas and nasopharangeal carcinomas ( Junker et al. (2005) J Urol. 173:2150-3 ; Sloan et al. (2004) Am J Pathol.
  • CD70 has been found to be overexpressed on T cells treated with DNA methyltransferase inhibitors or ERK pathway inhibitors, possibly leading to drug-induced and idiopathic lupus ( Oelke et al. (2004) Arthritis Rheum. 50:1850-60 ).
  • CD70 The interaction of CD70 with CD27 has also been proposed to play a role in cell-mediated autoimmune disease and the inhibition of TNF-alpha production ( Nakajima et al. (2000) J. Neuroimmunol. 109:188-96 ).
  • CD70 represents a valuable target for the treatment of cancer, autoimmune disorders and a variety of other diseases characterized by CD70 expression.
  • the present disclosure provides isolated monoclonal antibodies, in particular human monoclonal antibodies, that bind to CD70 and that exhibit numerous desirable properties. These properties include high affinity binding to human CD70. Also provided are methods for treating a variety CD70 mediated diseases using the antibodies and compositions of the instant disclosure.
  • the antibodies can be, for example, full-length antibodies, for example of an IgG1 or IgG4 isotype.
  • the antibodies can be antibody fragments, such as Fab or Fab' 2 fragments or single chain antibodies.
  • the immunoconjugate may comprise an antibody or antigen-binding portion thereof, of the invention, linked to a therapeutic agent, such as a cytotoxin or a radioactive isotope.
  • nucleic acid molecules encoding the antibodies or antigen-binding portions thereof, of the invention also provided are expression vectors comprising such nucleic acids, host cells comprising such expression vectors and methods for making anti-CD70 antibodies using such host cells.
  • the disclosure provides a transgenic mouse comprising human immunoglobulin heavy and light chain transgenes, wherein the mouse expresses an antibody of the invention, as well as hybridomas prepared from such a mouse, wherein the hybridoma produces the antibody of the invention.
  • the disease can be a cancer, e.g., renal cell carcinoma cancer or lymphoma.
  • the present disclosure relates to isolated monoclonal antibodies, particularly human monoclonal antibodies, that bind specifically to CD70 with high affinity.
  • the antibodies disclosed herein are derived from particular heavy and light chain germline sequences and/or comprise particular structural features such as CDR regions comprising particular amino acid sequences.
  • This disclosure describes isolated antibodies, methods of making such antibodies, immunoconjugates and bispecific molecules comprising such antibodies and pharmaceutical compositions containing the antibodies, immunconjugates or bispecific molecules of the disclosure.
  • This disclosure also describes methods of using the antibodies, such as to treat diseases such as cancer.
  • a “signal transduction pathway” refers to the biochemical relationship between a variety of signal transduction molecules that play a role in the transmission of a signal from one portion of a cell to another portion of a cell.
  • the phrase "cell surface receptor” includes, for example, molecules and complexes of molecules capable of receiving a signal and the transmission of such a signal across the plasma membrane of a cell.
  • An example of a “cell surface receptor” of the present disclosure is the CD70 receptor.
  • antibody as referred to herein includes whole antibodies and any antigen binding fragment (i.e., "antigen-binding portion") or single chains thereof.
  • An “antibody” refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds or an antigen binding portion thereof.
  • Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as V H ) and a heavy chain constant region.
  • the heavy chain constant region is comprised of three domains, C H1 , C H2 and C H3 .
  • Each light chain is comprised of a light chain variable region (abbreviated herein as V L ) and a light chain constant region.
  • the light chain constant region is comprised of one domain, C L .
  • the V H and V L regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR).
  • CDR complementarity determining regions
  • FR framework regions
  • Each V H and V L is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.
  • the constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g ., effector cells) and the first component (Clq) of the classical complement system.
  • antigen-binding portion of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e . g ., CD70). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.
  • binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the V L , V H , C L and C H1 domains; (ii) a F(ab') 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fab' fragment, which is essentially an Fab with part of the hinge region (see, Fundamental Immunology (Paul ed., 3rd ed.
  • the two domains of the Fv fragment, V L and V H are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the V L and V H regions pair to form monovalent molecules (known as single chain Fv (scFv); see, e.g., Bird et al. (1988) Science 242:423-426 ; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883 ).
  • Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody.
  • These antibody fragments are obtained using conventional techniques known to those with skill in the art and the fragments are screened for utility in the same manner as are intact antibodies.
  • an "isolated antibody”, as used herein, is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g ., an isolated antibody that specifically binds CD70 is substantially free of antibodies that specifically bind antigens other than CD70).
  • An isolated antibody that specifically binds CD70 may, however, have cross-reactivity to other antigens, such as CD70 molecules from other species.
  • an isolated antibody specifically binds to human CD70 and does not cross-react with other non-human CD70 antigens.
  • an isolated antibody may be substantially free of other cellular material and/or chemicals.
  • monoclonal antibody or “monoclonal antibody composition” as used herein refer to a preparation of antibody molecules of single molecular composition.
  • a monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.
  • human antibody is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences.
  • the human antibodies may include later modifications, including natural or synthetic modifications.
  • the human antibodies of the disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences ( e.g ., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).
  • the term "human antibody,” as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
  • human monoclonal antibody refers to antibodies displaying a single binding specificity which have variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences.
  • the human monoclonal antibodies are produced by a hybridoma which includes a B cell obtained from a transgenic nonhuman animal, e.g ., a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell.
  • recombinant human antibody includes all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from an animal (e.g ., a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom (described further below), (b) antibodies isolated from a host cell transformed to express the human antibody, e.g ., from a transfectoma, (c) antibodies isolated from a recombinant, combinatorial human antibody library and (d) antibodies prepared, expressed, created or isolated by any other means that involve splicing of human immunoglobulin gene sequences to other DNA sequences.
  • Such recombinant human antibodies have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences.
  • such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the V H and V L regions of the recombinant antibodies are sequences that, while derived from and related to human germline V H and V L sequences, may not naturally exist within the human antibody germline repertoire in vivo.
  • isotype refers to the antibody class (e.g., IgM or IgG1) that is encoded by the heavy chain constant region genes.
  • an antibody recognizing an antigen and "an antibody specific for an antigen” are used interchangeably herein with the term “an antibody which binds specifically to an antigen.”
  • human antibody derivatives refers to any modified form of the human antibody, e.g ., a conjugate of the antibody and another agent or antibody.
  • humanized antibody is intended to refer to antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. Additional framework region modifications may be made within the human framework sequences.
  • chimeric antibody is intended to refer to antibodies in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as an antibody in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody.
  • an antibody that "specifically binds to human CD70" is intended to refer to an antibody that binds to human CD70 with a K D of 5 x 10 -8 M or less, more preferably 1 x 10 -8 M or less, more preferably 6 x 10 -9 M or less, more preferably 3 x 10 -9 M or less, even more preferably 2 x 10 -9 M or less.
  • K assoc or "K a ", as used herein, is intended to refer to the association rate of a particular antibody-antigen interaction
  • K dis or “K a ,” as used herein, is intended to refer to the dissociation rate of a particular antibody-antigen interaction
  • K D is intended to refer to the dissociation constant, which is obtained from the ratio of K d to K a ( i.e ., K d /K a ) and is expressed as a molar concentration (M).
  • K D values for antibodies can be determined using methods well established in the art. A preferred method for determining the K D of an antibody is by using surface plasmon resonance, preferably using a biosensor system such as a Biacore® system.
  • high affinity for an IgG antibody refers to an antibody having a K D of 10 -7 M or less, more preferably 10 -8 M or less, more preferably 10 -9 M or less, and even more preferably 10 -10 M or less for a target antigen.
  • “high affinity” binding can vary for other antibody isotypes.
  • “high affinity” binding for 6an IgM isotype refers to an antibody having a K D of 10 -7 M or less, more preferably 10 -8 M or less, even more preferably 10 -9 M or less.
  • the term “subject” includes any human or nonhuman animal.
  • nonhuman animal includes all vertebrates, e.g. , mammals and non-mammals, such as nonhuman primates, sheep, dogs, cats, horses, cows, chickens, amphibians, fish, reptiles, etc.
  • the antibodies disclosed herein are characterized by particular functional features or properties of the antibodies.
  • the antibodies bind specifically to human CD70.
  • an antibody as disclosed herein binds to CD70 with high affinity, for example with a K D of 5 x 10 -7 M or less, even more preferably 5.5x 10 -9 or less, even more preferably 3x10 -9 or less, even more preferably 2x10 -9 or less or even more preferably 1.5x10 -9 or less.
  • Standard assays to evaluate the binding ability of the antibodies toward CD70 are known in the art, including for example, ELISAs, Western blots and RIAs. Suitable assays are described in detail in the Examples.
  • the binding kinetics (e.g., binding affinity) of the antibodies also can be assessed by standard assays known in the art, such as by ELISA, Scatchard and Biacore analysis.
  • the antibodies may bind to a renal carcinoma tumor cell line, for example, the 786-O, A-498, ACHN, Caki-1 or Caki-2 cell lines.
  • the antibodies may bind to a B-cell tumor cell line, for example, the Daudi, HuT 78, Raji or Granta-519 cell lines.
  • Exemplified antibodies include the human monoclonal antibodies 2H5, 10B4, 8B5, 18E7 and 69A7, isolated and structurally characterized as described in Examples 1 and 2.
  • the V H amino acid sequences of 2H5, 10B4, 8B5, 18E7 and 69A7 are shown in SEQ ID NOs: 1, 2, 3, 4 and 5, respectively.
  • the V L amino acid sequences of 2H5, 10B4, 8B5, 18E7 and 69A7 are shown in SEQ ID NOs: 6, 7, 8, 9 and 10, respectively.
  • a preferred heavy and light chain combination is:
  • antibodies that comprise the heavy chain and light chain CDR1s, CDR2s and CDR3s of 2H5, 10B4, 8B5, 18E7 and 69A7 or combinations thereof.
  • the amino acid sequences of the V H CDR1s of 2H5, 10B4, 8B5, 18E7 and 69A7 are shown in SEQ ID NOs: 11, 12, 13, 14 and 15, respectively.
  • the amino acid sequences of the V H CDR2s of 2H5, 10B4, 8B5, 18E7 and 69A7 are shown in SEQ ID NOs: 16, 17, 18, 19 and 20, respectively.
  • the amino acid sequences of the VH CDR3S of 2H5, 10B4, 8B5, 18E7 and 69A7 are shown in SEQ ID NOs: 21, 22, 23, 24 and 25, respectively.
  • the amino acid sequences of the V K CDR1s of 2H5, 10B4, 8B5, 18E7 and 69A7 are shown in SEQ ID NOs: 26, 27, 28, 29 and 30, respectively.
  • the amino acid sequences of the V K CDR2s of 2H5, 10B4, 8B5, 18E7 and 69A7 are shown in SEQ ID NOs: 31, 32, 33, 34 and 35, respectively.
  • the amino acid sequences of the V K CDR3s of 2H5, 10B4, 8B5, 18E7 and 69A7 are shown in SEQ ID NOs:36, 37, 38, 39 and 40, respectively.
  • the CDR regions are delineated using the Kabat system ( Kabat, E. A., et al (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242 ).
  • an isolated monoclonal antibody or antigen binding portion thereof comprising:
  • the antibody specifically binds CD70, preferably human CD70. Also disclosed herein is an antibody which comprises:
  • antibodies which comprise a heavy chain variable region from a particular germline heavy chain immunoglobulin gene and/or a light chain variable region from a particular germline light chain immunoglobulin gene.
  • an isolated monoclonal antibody or an antigen-binding portion thereof comprising a heavy chain variable region that is the product of or derived from a human V H 3-30.3 gene, wherein the antibody specifically binds CD70.
  • an isolated monoclonal antibody or an antigen-binding portion thereof comprising a heavy chain variable region that is the product of or derived from a human V H 3-33 gene, wherein the antibody specifically binds CD70.
  • an isolated monoclonal antibody or an antigen-binding portion thereof comprising a heavy chain variable region that is the product of or derived from a human V H 4-61 gene, wherein the antibody specifically binds CD70.
  • an isolated monoclonal antibody or an antigen-binding portion thereof comprising a light chain variable region that is the product of or derived from a human V K L6 gene, wherein the antibody specifically binds CD70. Also disclosed is an isolated monoclonal antibody or an antigen-binding portion thereof, comprising a light chain variable region that is the product of or derived from a human V K L18 gene, wherein the antibody specifically binds CD70. Also disclosed is an isolated monoclonal antibody or an antigen-binding portion thereof, comprising a light chain variable region that is the product of or derived from a human V K L15 gene, wherein the antibody specifically binds CD70.
  • An example of an antibody having V H and V K of V H 3-30.3 and V K L6, respectively, is 2H5.
  • An example of an antibody having V H and V K of V H 3-30.3 and V K L18, respectively, is 10B4.
  • Examples of antibodies having V H and V K of V H 3-33 and V K L15, respectively, are 8B5 and 18E7.
  • An example of an antibody having V H and V K of V H 4-61 and V K L6, respectively, is 69A7.
  • a human antibody comprises heavy or light chain valuable regions that is "the product of” or “derived from” a particular germline sequence if the variable regions of the antibody are obtained from a system that uses human germline immunoglobulin genes, Such systems include immunizing a transgenic mouse carrying human immunoglobulin genes with the antigen of interest or screening a human immunoglobulin gene library displayed on phage with the antigen of interest.
  • a human antibody that is "the product of” or “derived from” a human germline immunoglobulin sequence can be identified as such by comparing the amino acid sequence of the human antibody to the amino acid sequences of human germline immunoglobulins and selecting the human germline immunoglobulin sequence that is closest in sequence (i.e., greatest % identity) to the sequence of the human antibody.
  • a human antibody that is "the product of” or “derived from” a particular human germline immunoglobulin sequence may contain amino acid differences as compared to the germline sequence, due to, for example, naturally-occurring somatic mutations or intentional introduction of site-directed mutation.
  • a selected human antibody typically is at least 90% identical in amino acids sequence to an amino acid sequence encoded by a human germline immunoglobulin gene and contains amino acid residues that identify the human antibody as being human when compared to the germline immunoglobulin amino acid sequences of other species (e.g., murine germline sequences).
  • a human antibody may be at least 95% or even at least 96%, 97%, 98% or 99% identical in amino acid sequence to the amino acid sequence encoded by the germline immunoglobulin gene.
  • a human antibody derived from a particular human germline sequence will display no more than 10 amino acid differences from the amino acid sequence encoded by the human germline immunoglobulin gene.
  • the human antibody may display no more than 5 or even no more than 4, 3, 2 or 1 amino acid difference from the amino acid sequence encoded by the germline immunoglobulin gene.
  • an antibody that comprises heavy and light chain variable regions comprising amino acid sequences that are homologous to the amino acid sequences of the preferred antibodies described herein and wherein the antibodies retain the desired functional properties of the anti-CD70 antibodies of the disclosure.
  • the antibody specifically binds to CD70.
  • the percent homology between two amino acid sequences is equivalent to the percent identity between the two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the non-limiting examples below.
  • the percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput, Appl. Biosci., 4: 11-17 (1988 )) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J Mol. Biol.
  • the protein sequences of the present disclosure can further be used as a "query sequence" to perform a search against public databases to, for example, identify related sequences.
  • Such searches can be performed using the XBLAST program (version 2.0) of Altschul, et al (1990) J. Mol. Biol. 215:403-10 .
  • Gapped BLAST can be utilized as described in Altschul et al, (1997) Nucleic Acids Res. 25(17):33389-3402 .
  • the default parameters of the respective programs e.g. , XBLAST and NBLAST
  • the default parameters of the respective programs e.g. , XBLAST and NBLAST
  • antibodies that bind to the same epitope on human CD70 as any of the CD70 monoclonal antibodies of the invention i.e., antibodies that have the ability to cross-compete for binding to CD70 with any of the monoclonal antibodies of the invention.
  • the reference antibody for cross-competition studies can be the monoclonal antibody 2H5 (having V H and V L sequences as shown in SEQ ID NOs: 1 and 6, respectively).
  • Such cross-competing antibodies can be identified based on their ability to cross-compete with 2H5 in standard CD70 binding assays. For example, BIAcore analysis, ELISA assays or flow cytometry may be used to demonstrate cross-competition with the antibodies of the current disclosure.
  • test antibody to inhibit the binding of, for example, 2H5 to human CD70 demonstrates that the test antibody can compete with 2H5 for binding to human CD70 and thus binds to the same epitope on human CD70 as 2H5.
  • the antibody that binds to the same epitope on human CD70 as 2H5 is a human monoclonal antibody.
  • human monoclonal antibodies can be prepared and isolated as described in the Examples.
  • the antibodies disclosed herein may be further characterized by the various physical properties of the antibodies.
  • Various assays may be used to detect and/or differentiate different classes of antibodies based on these physical properties.
  • Antibodies disclosed herein may contain one or more glycosylation sites in either the light or heavy chain variable region.
  • the presence of one or more glycosylation sites in the variable region may result in increased immunogenicity of the antibody or an alteration of the pK of the antibody due to altered antigen binding ( Marshall et al (1972) Annu Rev Biochem 41:673-702 ; Gala FA and Morrison SL (2004) J Immunol 172:5489-94 ; Wallick et al (1988) JExp Med 168: 1099- 109 ; Spiro RG (2002) Glycobiology 12:43R-56R ; Parekh et al (1985) Nature 316:452-7 ; Mimura et al.
  • variable region glycosylation may be tested using a Glycoblot assay, which cleaves the antibody to produce a Fab, and then tests for glycosylation using an assay that measures periodate oxidation and Schiff base formation.
  • variable region glycosylation may be tested using Dionex light chromatography (Dionex-LC), which cleaves saccharides from a Fab into monosaccharides and analyzes the individual saccharide content.
  • Dionex-LC Dionex light chromatography
  • Antibodies disclosed herein may preferably not contain asparagine isomerism sites.
  • a deamidation or isoaspartic acid effect may occur on N-G or D-G sequences, respectively.
  • the deamidation or isoaspartic acid effect results in the creation of isoaspartic acid which decreases the stability of an antibody by creating a kinked structure off a side chain carboxy terminus rather than the main chain.
  • the creation of isoaspartic acid can be measured using an iso-quant assay, which uses a reverse-phase HPLC to test for isoaspartic acid.
  • Each antibody will have a unique isoelectric point (pI), but generally antibodies will fall in the pH range of between 6 and 9.5.
  • the pI for an IgG1 antibody typically falls within the pH range of 7-9.5 and the pI for an IgG4 antibody typically falls within the pH range of 6-8.
  • Antibodies may have a pI that is outside this range. Although the effects are generally unknown, there is speculation that antibodies with a pI outside the normal range may have some unfolding and instability under in vivo conditions.
  • the isoelectric point may be tested using a capillary isoelectric focusing assay, which creates a pH gradient and may utilize laser focusing for increased accuracy ( Janini et al (2002) Electrophoresis 23:1605-11 ; Ma et al (2001) Chromatographia 53:S75-89 ; Hunt et al (1998) J Chromatogr A 800:355-67 ).
  • Each antibody will have a melting temperature that is indicative of thermal stability ( Krishnamurthy R and Manning MC (2002) Curr Pharm Biotechnol 3:361-71 ).
  • a higher thermal stability indicates greater overall antibody stability in vivo.
  • the melting point of an antibody may be measure using techniques such as differential scanning calorimetry ( Chen et al (2003) Pharm Res 20: 1952-60 ; Ghirlando et al (1999) Immunol Lett 68:47-52 ).
  • T M1 indicates the temperature of the initial unfolding of the antibody.
  • T M2 indicates the temperature of complete unfolding of the antibody.
  • the T M1 of an antibody of the present disclosure is greater than 60°C, preferably greater than 65°C, even more preferably greater than 70°C.
  • the thermal stability of an antibody may be measure using circular dichroism ( Murray et al. (2002) J Chromatogr Sci 40:343-9 ).
  • Antibodies disclosed herein may be selected that do not rapidly degrade. Fragmentation of an anti-CD 19 antibody may be measured using capillary electrophoresis (CE) and MALDI-MS, as is well understood in the art ( Alexander AJ and Hughes DE (1995) Anal Chem 67:3626-32 ).
  • CE capillary electrophoresis
  • MALDI-MS MALDI-MS
  • Antibodies disclosed herein may be selected that have minimal aggregation effects. Aggregation may lead to triggering of an unwanted immune response and/or altered or unfavorable pharmacokinetic properties. Generally, antibodies are acceptable with aggregation of 25% or less, preferably 20% or less, even more preferably 15% or less, even more preferably 10% or less and even more preferably 5% or less. Aggregation may be measured by several techniques well known in the art, including size-exclusion column (SEC) high performance liquid chromatography (FIPLC), and light scattering to identify monomers, dimers, trimers or multimers.
  • SEC size-exclusion column
  • FIPLC high performance liquid chromatography
  • nucleic acid molecules that encode the antibodies of the disclosure.
  • the nucleic acids may be present in whole cells, in a cell lysate or in a partially purified or substantially pure form.
  • a nucleic acid is "isolated” or “rendered substantially pure” when purified away from other cellular components or other contaminants, e.g. , other cellular nucleic acids or proteins, by standard techniques, including alkaline/SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis and others well known in the art. See, F. Ausubel, et at, ed. (1987) Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York .
  • a nucleic acid can be, for example, DNA or RNA and may or may not contain intronic sequences.
  • the nucleic acid is a cDNA molecule.
  • Nucleic acids can be obtained using standard molecular biology techniques.
  • hybridomas e.g ., hybridomas prepared from transgenic mice carrying human immunoglobulin genes as described further below
  • cDNAs encoding the light and heavy chains of the antibody made by the hybridoma can be obtained by standard PCR amplification or cDNA cloning techniques.
  • nucleic acid encoding the antibody can be recovered from the library.
  • nucleic acids molecules encoding the VH and VL sequences of the 2H5, 10B4, 8B5, 18E7 or 69A7 monoclonal antibodies.
  • DNA sequences encoding the VH sequences of 2H5, 10B4, 8B5, 18E7 and 69A7 are shown in SEQ ID NOs:41, 42, 43, 44 and 45, respectively.
  • DNA sequences encoding the VL sequences of 2H5, 10B4, 8B5, 18E7 and 69A7 are shown in SEQ ID NOs:46, 47, 48, 49 and 50, respectively.
  • VH and VL segments are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example to convert the variable region genes to full-length antibody chain genes, to Fab fragment genes or to a scFv gene.
  • a VL- or VH-encoding DNA fragment is operatively linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker.
  • the term "operatively linked”, as used in this context, is intended to mean that the two DNA fragments are joined such that the amino acid sequences encoded by the two DNA fragments remain in-frame.
  • the isolated DNA encoding the VH region can be converted to a full-length heavy chain gene by operatively linking the VH-encoding DNA to another DNA molecule encoding heavy chain constant regions (CH1, CH2 and CH3).
  • heavy chain constant regions CH1, CH2 and CH3
  • the sequences of human heavy chain constant region genes are known in the art (see e.g., Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242 ) and DNA fragments encompassing these regions can be obtained by standard PCR amplification.
  • the heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but most preferably is an IgG1, IgG2, IgG3 or IgG4 constant region.
  • the VH-encoding DNA can be operatively linked to another DNA molecule encoding only the heavy chain CH1 constant region.
  • the isolated DNA encoding the VL region can be converted to a full-length light chain gene (as well as a Fab light chain gene) by operatively linking the VL-encoding DNA to another DNA molecule encoding the light chain constant region, CL.
  • the sequences of human light chain constant region genes are known in the art (see e.g., Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242 ) and DNA fragments encompassing these regions can be obtained by standard PCR amplification.
  • the light chain constant region can be a kappa or lambda constant region, but most preferably is a kappa constant region.
  • the VH- and VL-encoding DNA fragments are operatively linked to another fragment encoding a flexible linker, e.g., encoding the amino acid sequence (Gly4 -Ser) 3 , such that the VH and VL sequences can be expressed as a contiguous single-chain protein, with the VL and VH regions joined by the flexible linker (see e.g., Bird et al. (1988) Science 242:423-426 ; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883 ; McCafferty et al., (1990) Nature 348:552-554 ).
  • a flexible linker e.g., encoding the amino acid sequence (Gly4 -Ser) 3
  • Monoclonal antibodies can be produced by a variety of techniques, including conventional monoclonal antibody methodology e.g ., the standard somatic cell hybridization technique of Kohler and Milstein (1975) Nature 256:495 . Although somatic cell hybridization procedures are preferred, in principle, other techniques for producing monoclonal antibody can be employed e.g. , viral or oncogenic transformation of B lymphocytes.
  • the preferred animal system for preparing hybridomas is the murine system.
  • Hybridoma production in the mouse is a very well-established procedure. Immunization protocols and techniques for isolation of immunized splenocytes for fusion are known in the art, Fusion partners (e.g ., murine myeloma cells) and fusion procedures are also known.
  • Chimeric or humanized antibodies can be prepared based on the sequence of a murine monoclonal antibody prepared as described above.
  • DNA encoding the heavy and light chain immunoglobulins can be obtained from the murine hybridoma of interest and engineered to contain non-murine ( e.g ., human) immunoglobulin sequences using standard molecular biology techniques.
  • the murine variable regions can be linked to human constant regions using methods known in the art (see e.g., U.S. Patent No. 4,816,567 to Cabilly et al ).
  • the murine CDR regions can be inserted into a human framework using methods known in the art (see e.g., U.S. Patent No. 5,225,539 to Winter and U.S. Patent Nos. 5,530,101 ; 5,585,089 ; 5,693,762 and 6,180,370 to Queen et al ).
  • the antibodies of the invention are human monoclonal antibodies.
  • Such human monoclonal antibodies directed against CD70 can be generated using transgenic or transchromosomic mice carrying parts of the human immune system rather than the mouse system.
  • transgenic and transchromosomic mice include mice referred to herein as the HuMAb mouse and KM mouse®, respectively and are collectively referred to herein as "human Ig mice”.
  • the HuMAb mouse® (Medarex, Inc.) contains human immunoglobulin gene miniloci that encode unrearranged human heavy ( ⁇ and ⁇ ) and ⁇ light chain immunoglobulin sequences, together with targeted mutations that inactivate the endogenous ⁇ and ⁇ chain loci (see e.g., Lonberg, et al. (1994) Nature 368(6474): 856-859 ). Accordingly, the mice exhibit reduced expression of mouse IgM or ⁇ and in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to generate high affinity human IgG ⁇ monoclonal (Lonberg, N. et al. (1994), supra; reviewed in Lonberg, N.
  • Antibodies can be raised using a mouse that carries human immunoglobulin sequences on transgenes and transchomosomes, such as a mouse that carries a human heavy chain transgene and a human light chain transchromosome.
  • a mouse that carries a human heavy chain transgene and a human light chain transchromosome Such mice, referred to herein as the "KM mouse®", are described in detail in PCT Publication WO 02/43478 to Ishida et al.
  • transgenic animal systems expressing human immunoglobulin genes are available in the art and can be used to raise anti-CD70 antibodies.
  • an alternative transgenic system referred to as the Xenomouse (Abgenix, Inc.) can be used; such mice are described in, for example, U.S. Patent Nos. 5,939,598 ; 6,075,181 ; 6,114,598 ; 6, 150,584 and 6,162,963 to Kucherlapati et al.
  • mice carrying both a human heavy chain transchromosome and a human light chain tranchromosome referred to as "TC mice” can be used; such mice are described in Tomizuka et al (2000) Proc. Natl. Acad. Sci. USA 97:722-727 .
  • cows carrying human heavy and light chain transchromosomes have been described in the art ( Kuroiwa et al. (2002) Nature Biotechnology 20:889-894 ) and can be used to raise anti-CD70 antibodies.
  • Human monoclonal antibodies can also be prepared using phage display methods for screening libraries of human immunoglobulin genes. Such phage display methods for isolating human antibodies are established in the art. See for example: U.S. Patent Nos. 5,223,409 ; 5,403,484 ; and 5,571,698 to Ladner et al ; U.S. Patent Nos. 5,427,908 and 5,580,717 to Dower et al ; U.S. Patent Nos. 5,969,108 and 6,172,197 to McCafferty et al ; and U.S. Patent Nos. 5,885,793 ; 6,521,404 ; 6,544,731 ; 6,555,313 ; 6,582,915 and 6,593,081 to Griffiths et al.
  • Human monoclonal antibodies can also be prepared using SCID mice into which human immune cells have been reconstituted such that a human antibody response can be generated upon immunization.
  • SCID mice into which human immune cells have been reconstituted such that a human antibody response can be generated upon immunization.
  • Such mice are described in, for example, U.S. Patent Nos. 5,476,996 and 5,698,767 to Wilson et al.
  • mice When human Ig mice are used to raise human antibodies, such mice can be immunized with a CD70-expressing cell line, a purified or enriched preparation of CD70 antigen and/or recombinant CD70 or an CD70 fusion protein, as described by Lonberg, N. et al. (1994) Nature 368(6474): 856-859 ; Fishwild, D. et al (1996) Nature Biotechnology 14: 845-851 ; and PCT Publication WO 98/24884 and WO 01/14424 .
  • the mice will be 6-16 weeks of age upon the first immunization.
  • a purified or recombinant preparation (5-50 ⁇ g) of CD70 antigen can be used to immunize the human Ig mice intraperitoneally.
  • Example 1 Cumulative experience with various antigens has shown that the transgenic mice respond when initially immunized intraperitoneally (IP) with antigen in complete Freund's adjuvant, followed by every other week IP immunizations up to a total of 6 with antigen in incomplete Freund's adjuvant. However, adjuvants other than Freund's are also found to be effective. In addition, whole cells in the absence of adjuvant are found to be highly immunogenic. The immune response can be monitored over the course of the immunization protocol with plasma samples being obtained, for example, by retroorbital bleeds.
  • the plasma can be screened by ELISA and mice with sufficient titers of anti-CD70 human immunoglobulin can be used for fusions (as described in Example 1). Mice can be boosted intravenously with antigen 3 days before sacrifice and removal of the spleen. It is expected that 2-3 fusions for each immunization may need to be performed. Between 6 and 24 mice are typically immunized for each antigen. Usually both HCo7 and HCo12 strains are used. Generation of HCo7 and HCo12 mouse strains are described in U.S. Patent No. 5,770,429 and Example 2 of PCT Publication WO 01/09187 , respectively.
  • both HCo7 and HCo12 transgene can be bred together into a single mouse having two different human heavy chain transgenes (HCo7/HCo12).
  • the KM mouse® strain can be used, as described in PCT Publication WO 02/43478 .
  • splenocytes and/or lymph node cells from immunized mice can be isolated and fused to an appropriate immortalized cell line, such as a mouse myeloma cell line.
  • an appropriate immortalized cell line such as a mouse myeloma cell line.
  • the resulting hybridomas can be screened for the production of antigen-specific antibodies.
  • single cell suspensions of splenic lymphocytes from immunized mice can be fused to one-sixth the number of P3X63-Ag8.653 nonsecreting mouse myeloma cells (ATCC, CRL 1580) with 50% PEG.
  • the single cell suspensions of splenic lymphocytes from immunized mice can be fused using an electric field based electrofusion method, using a Cyto Pulse large chamber cell fusion electroporator (Cyto Pulse Sciences, Inc., Glen Burnie, MD).
  • Cells are plated at approximately 2 x 10 5 in flat bottom microtiter plate, followed by a one week incubation in DMEM high glucose medium with L-glutamine and sodium pyruvate (Mediatech, Inc., Herndon, VA) and further containing 20% fetal Bovine Serum (Hyclone, Logan, UT), 18% P388DI conditional media, 5% Origen Hybridoma cloning factor (BioVeris, Gaithersburg, VA), 4 mM L-glutamine, 5mM HEPES, 0.055 mM ⁇ -mercaptoethanol, 50 units/ml penicillin, 50 mg/ml streptomycin and IX Hypoxanthine-aminopterin-thymidine (HAT) media (Sigma; the HAT is added 24 hours after the fusion).
  • DMEM high glucose medium with L-glutamine and sodium pyruvate Mediatech, Inc., Herndon, VA
  • 20% fetal Bovine Serum Hyclon
  • selected hybridomas can be grown in two-liter spinner-flasks for monoclonal antibody purification.
  • Supernatants can be filtered and concentrated before affinity chromatography with protein A-sepharose (Pharmacia, Piscataway, N. J.).
  • Eluted IgG can be checked by gel electrophoresis and high performance liquid chromatography to ensure purity.
  • the buffer solution can be exchanged into PBS and the concentration can be determined by OD280 using 1.43 extinction coefficient.
  • the monoclonal antibodies can be aliquoted and stored at -80° C.
  • Antibodies can be produced in a host cell transfectoma using, for example, a combination of recombinant DNA techniques and gene transfection methods as is well known in the art (e.g. , Morrison, S. (1985) Science 229:1202 ).
  • DNAs encoding partial or full-length light and heavy chains can be obtained by standard molecular biology techniques (e.g. , PCR amplification or cDNA cloning using a hybridoma that expresses the antibody of interest) and the DNAs can be inserted into expression vectors such that the genes are operatively linked to transcriptional and translational control sequences.
  • operatively linked is intended to mean that an antibody gene is ligated into a vector such that transcriptional and translational control sequences within the vector serve their intended function of regulating the transcription and translation of the antibody gene.
  • the expression vector and expression control sequences are chosen to be compatible with the expression host cell used.
  • the antibody light chain gene and the antibody heavy chain gene can be inserted into separate vector or, more typically, both genes are inserted into the same expression vector.
  • the antibody genes are inserted into the expression vector by standard methods (e.g. , ligation of complementary restriction sites on the antibody gene fragment and vector or blunt end ligation if no restriction sites are present).
  • the light and heavy chain variable regions of the antibodies described herein can be used to create full-length antibody genes of any antibody isotype by inserting them into expression vectors already encoding heavy chain constant and light chain constant regions of the desired isotype such that the V H segment is operatively linked to the C H segment(s) within the vector and the V K segment is operatively linked to the C L segment within the vector.
  • the recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody chain from a host cell.
  • the antibody chain gene can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene.
  • the signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).
  • the recombinant expression vectors of the disclosure carry regulatory sequences that control the expression of the antibody chain genes in a host cell.
  • the term "regulatory sequence” is intended to include promoters, enhancers and other expression control elements (e.g. , polyadenylation signals) that control the transcription or translation of the antibody chain genes.
  • Such regulatory sequences are described, for example, in Goeddel (Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, CA (1990 )). It will be appreciated by those skilled in the art that the design of the expression vector, including the selection of regulatory sequences, may depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc.
  • Preferred regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from cytomegalovirus (CMV), Simian Virus 40 (SV40), adenovirus, ( e.g ., the adenovirus major late promoter (AdMLP) and polyoma.
  • CMV cytomegalovirus
  • SV40 Simian Virus 40
  • AdMLP adenovirus major late promoter
  • nonviral regulatory sequences may be used, such as the ubiquitin promoter or ⁇ -globin promoter.
  • regulatory elements composed of sequences from different sources such as the SR ⁇ promoter system, which contains sequences from the SV40 early promoter and the long terminal repeat of human T cell leukemia virus type 1 ( Takebe, Y. et al. (1988) Mol. Cell. Biol. 8:466-472 ).
  • the recombinant expression vectors of the disclosure may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g. origins of replication) and selectable marker genes.
  • the selectable marker gene facilitates selection of host cells into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216 , 4,634,665 and 5,179,017, all by Axel et al. ) .
  • the selectable marker gene confers resistance to drugs, such as G418, hygromycin or methotrexate, on a host cell into which the vector has been introduced.
  • Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr- host cells with methotrexate selection/amplification) and the neo gene (for G418 selection).
  • DHFR dihydrofolate reductase
  • the expression vector(s) encoding the heavy and light chains is transfected into a host cell by standard techniques.
  • the various forms of the term "transfection" are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, e.g ., electroporation, calcium-phosphate precipitation, DEAE-dextran transfection and the like.
  • Preferred mammalian host cells for expressing recombinant antibodies include Chinese Hamster Ovary (CHO cells) (including dhfr- CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci USA 77:4216-4220 , used with a DHFR selectable marker, e.g., as described in R. J. Kaufman and P. A. Sharp (1982) Mol Biol. 159:601-621 ), NSO myeloma cells, COS cells and SP2 cells.
  • Chinese Hamster Ovary CHO cells
  • dhfr- CHO cells described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci USA 77:4216-4220 , used with a DHFR selectable marker, e.g., as described in R. J. Kaufman and P. A. Sharp (1982) Mol Biol. 159:601-621
  • NSO myeloma cells COS cells and SP
  • another preferred expression system is the GS gene expression system disclosed in WO 87/04462 , WO 89/01036 and EP 338,841 .
  • the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or, more preferably, secretion of the antibody into the culture medium in which the host cells are grown.
  • Antibodies can be recovered from the culture medium using standard protein purification methods.
  • Antibodies can be tested for binding to CD70 by, for example, flow cytometry. Briefly, CD70-expressing cells are freshly harvested from tissue culture flasks and a single cell suspension prepared. CD70-expressing cell suspensions are either stained with primary antibody directly or after fixation with 1% paraformaldehyde in PBS. Approximately one million cells are resuspended in PBS containing 0.5% BSA and 50-200 ⁇ g/ml of primary antibody and incubated on ice for 30 minutes.
  • the cells are washed twice with PBS containing 0.1% BSA, 0.01% NaN3, resuspended in 100 ⁇ l of 1:100 diluted FITC-conjugated goat-anti-human IgG (Jackson ImmunoResearch, West Grove, PA) and incubated on ice for an additional 30 minutes.
  • the cells are again washed twice, resuspended in 0.5 ml of wash buffer and analyzed for fluorescent staining on a FACSCalibur cytometer (Becton-Dickinson, San Jose, CA).
  • antibodies can be tested for binding to CD70 by standard ELISA. Briefly, microtiter plates are coated with purified CD70 at 0.25 ⁇ g/ml in PBS and then blocked with 5% bovine serum albumin in PBS. Dilutions of antibody (e.g., dilutions of plasma from CD70-immunized mice) are added to each well and incubated for 1-2 hours at 37°C. The plates are washed with PBS/Tween and then incubated with secondary reagent (e.g., for human antibodies, a goat-anti-human IgG Fc-specific polyclonal reagent) conjugated to alkaline phosphatase for 1 hour at 37°C. After washing, the plates are developed with pNPP substrate (1 mg/ml) and analyzed at OD of 405-650. Preferably, mice which develop the highest titers will be used for fusions.
  • secondary reagent e.g., for human antibodies, a goat-anti-human I
  • An ELISA assay as described above can also be used to screen for hybridomas that show positive reactivity with CD70 immunogen.
  • Hybridomas that bind with high avidity to CD70 are subcloned and further characterized.
  • One clone from each hybridoma, which retains the reactivity of the parent cells (by ELISA) can be chosen for making a 5-10 vial cell bank stored at -140°C and for antibody purification.
  • selected hybridomas can be grown in two-liter spinner-flasks for monoclonal antibody purification.
  • Supernatants can be filtered and concentrated before affinity chromatography with protein A-sepharose (Pharmacia, Piscataway, NJ).
  • Eluted IgG can be checked by gel electrophoresis and high performance liquid chromatography to ensure purity.
  • the buffer solution can be exchanged into PBS and the concentration can be determined by OD280 using 1.43 extinction coefficient.
  • the monoclonal antibodies can be aliquoted and stored at -80°C.
  • each antibody can be biotinylated using commercially available reagents (Pierce, Rockford, IL). Competition studies using unlabeled monoclonal antibodies and biotinylated monoclonal antibodies can be performed using CD70 coated-ELISA plates as described above. Biotinylated mAb binding can be detected with a strep-avidin-alkaline phosphatase probe. Alternatively, competition studies can be performed using radiolabeled antibody and unlabelled competing antibodies can be detected in a Scatchard analysis, as further described in the Examples below.
  • isotype ELISAs can be performed using reagents specific for antibodies of a particular isotype. For example, to determine the isotype of a human monoclonal antibody, wells of microtiter plates can be coated with 1 ⁇ g/ml of anti-human immunoglobulin overnight at 4°C. After blocking with 1% BSA, the plates are reacted with 1 ⁇ g /ml or less of test monoclonal antibodies or purified isotype controls, at ambient temperature for one to two hours. The wells can then be reacted with either human IgG1 or human IgM-specific alkaline phosphatase-conjugated probes. Plates are developed and analyzed as described above.
  • Anti-CD70 human IgGs can be further tested for reactivity with CD70 antigen by Western blotting. Briefly, CD70 can be prepared and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. After electrophoresis, the separated antigens are transferred to nitrocellulose membranes, blocked with 10% fetal calf serum and probed with the monoclonal antibodies to be tested. Human IgG binding can be detected using anti-human IgG alkaline phosphatase and developed with BCIP/NBT substrate tablets (Sigma Chem. Co., St. Louis, Mo.).
  • an anti-CD70 antibody or a fragment thereof, conjugated to a therapeutic moiety such as a cytotoxin, a drug (e.g., an immunosuppressant) or a radiotoxin.
  • a therapeutic moiety such as a cytotoxin, a drug (e.g., an immunosuppressant) or a radiotoxin.
  • conjugates are referred to herein as “immunoconjugates”.
  • Immunoconjugates that include one or more cytotoxins are referred to as "immunotoxins”.
  • a cytotoxin or cytotoxic agent includes any agent that is detrimental to (e.g., kills) cells.
  • Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol and puromycin and analogs or homologs thereof.
  • Therapeutic agents also include, for example, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thiotepa, chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g ., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics ( e.g ., dactinomycin (formerly actinomycin), bleomycin, mithramycin and anthramycin (AMC)) and anti-mitotic agents ( e.
  • An example of a calicheamicin antibody conjugate is commercially available (MylotargTM; Wyeth-Ayerst).
  • Cytoxins can be conjugated to antibodies using linker technology available in the art.
  • linker types that have been used to conjugate a cytotoxin to an antibody include, but are not limited to, hydrazones, thioethers, esters, disulfides and peptide-containing linkers.
  • a linker can be chosen that is, for example, susceptible to cleavage by low pH within the lysosomal compartment or susceptible to cleavage by proteases, such as proteases preferentially expressed in tumor tissue such as cathepsins ( e.g ., cathepsins B, C, D).
  • Antibodies can also be conjugated to a radioactive isotope to generate cytotoxic radiopharmaceuticals, also referred to as radioimmunoconjugates.
  • radioactive isotopes that can be conjugated to antibodies for use diagnostically or therapeutically include, but are not limited to, iodine 131 , iodine 125 , indium 111 , yttrium 90 and lutetium Method for preparing radioimmunconjugates are established in the art.
  • Examples of radioimmunoconjugates are commercially available, including ZevalinTM (IDEC Pharmaceuticals) and BexxarTM (Corixa Pharmaceuticals) and similar methods can be used to prepare radioimmunoconjugates using the antibodies of the disclosure.
  • Antibody conjugates can be used to modify a given biological response and the drug moiety is not to be construed as limited to classical chemical therapeutic agents.
  • the drug moiety may be a protein or polypeptide possessing a desired biological activity.
  • proteins may include, for example, an enzymatically active toxin or active fragment thereof, such as abrin, ricin A, pseudomonas exotoxin or diphtheria toxin; a protein such as tumor necrosis factor or interferon- ⁇ ; or, biological response modifiers such as, for example, lymphokines, interleukin-1 ("IL-1"), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colony stimulating factor (“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”) or other growth factors.
  • IL-1 interleukin-1
  • IL-2 interleukin-2
  • IL-6 interleukin-6
  • GM-CSF granulocyte macrophag
  • compositions e.g ., a pharmaceutical composition, containing one or a combination of monoclonal antibodies or antigen-binding portion(s) thereof, of the invention, formulated together with a pharmaceutically acceptable carrier.
  • Such compositions may include one or a combination of (e.g., two or more different) antibodies or immunoconjugates of the invention.
  • a pharmaceutical composition can comprise a combination of antibodies (or immunoconjugates) that have complementary activities.
  • compositions can be administered in combination therapy, i.e., combined with other agents.
  • the combination therapy can include an anti-CD70 antibody of the invention combined with at least one other anti-inflammatory or immunosuppressant agent. Examples of therapeutic agents that can be used in combination therapy are described in greater detail below in the section on uses of the antibodies of the disclosure.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like that are physiologically compatible.
  • the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration ( e.g ., by injection or infusion).
  • the active compound i.e., antibody or immunoconjuage
  • the pharmaceutical compounds may include one or more pharmaceutically acceptable salts.
  • a "pharmaceutically acceptable salt” refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects (see e.g., Berge, S. M., et al. (1977) J. Pharm. Sci. 66:1-19 ). Examples of such salts include acid addition salts and base addition salts.
  • Acid addition salts include those derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like, as well as from nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like.
  • nontoxic inorganic acids such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like
  • nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like.
  • Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium and the like, as well as from nontoxic organic amines, such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine and the like.
  • a pharmaceutical composition may include a pharmaceutically acceptable antioxidant.
  • pharmaceutically acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid and the like.
  • water soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like
  • oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), buty
  • aqueous and nonaqueous carriers examples include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like) and suitable mixtures thereof, vegetable oils, such as olive oil and injectable organic esters, such as ethyl oleate.
  • polyols such as glycerol, propylene glycol, polyethylene glycol and the like
  • vegetable oils such as olive oil
  • injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants.
  • compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of microorganisms may be ensured both by sterilization procedures, supra and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
  • Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • the use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of the disclosure is contemplated. Supplementary active compounds can also be incorporated into the compositions.
  • compositions typically must be sterile and stable under the conditions of manufacture and storage.
  • the composition can be formulated as a solution, microemulsion, liposome or other ordered structure suitable to high drug concentration.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol and the like) and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol or sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the subject being treated and the particular mode of administration.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the composition which produces a therapeutic effect. Generally, out of one hundred per cent, this amount will range from about 0.01 per cent to about ninety-nine percent of active ingredient, preferably from about 0.1 per cent to about 70 per cent, most preferably from about 1 per cent to about 30 per cent of active ingredient in combination with a pharmaceutically acceptable carrier.
  • Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.
  • Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the disclosure are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.
  • the dosage ranges from about 0.0001 to 100 mg/kg and more usually 0.01 to 25 mg/kg, of the host body weight.
  • dosages can be 0.3 mg/kg body weight, 1 mg/kg body weight, 3 mg/kg body weight, 5 mg/kg body weight or 10 mg/kg body weight or within the range of 1-10 mg/kg.
  • Higher dosages e.g., 15 mg/kg body weight, 20 mg/kg body weight or 25 mg/kg body weight can be used as needed.
  • An exemplary treatment regime entails administration once per week, once every two weeks, once every three weeks, once every four weeks, once a month, once every 3 months or once every three to 6 months.
  • Particular dosage regimens for an anti-CD70 antibody of the disclosure include 1 mg/kg body weight or 3 mg/kg body weight via intravenous administration, with the antibody being given using one of the following dosing schedules: (i) every four weeks for six dosages, then every three months; (ii) every three weeks; (iii) 3 mg/kg body weight once followed by 1 mg/kg body weight every three weeks.
  • two or more anti-CD70 monoclonal antibodies with different binding specificities are administered simultaneously, in which case the dosage of each antibody administered falls within the ranges indicated.
  • Antibody is usually administered on multiple occasions. Intervals between single dosages can be, for example, weekly, monthly, every three months or yearly. Intervals can also be irregular as indicated by measuring blood levels of antibody to the target antigen in the patient. In some methods, dosage is adjusted to achieve a plasma antibody concentration of about 1-1000 ⁇ g /ml and in some methods about 25-300 ⁇ g /ml.
  • antibody can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the antibody in the patient. In general, human antibodies show the longest half life, followed by humanized antibodies, chimeric antibodies and nonhuman antibodies. The dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a relatively low dosage is administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated and preferably until the patient shows partial or complete amelioration of symptoms of disease. Thereafter, the patient can be administered a prophylactic regime.
  • Actual dosage levels of the active ingredients in the pharmaceutical compositions may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition and mode of administration, without being toxic to the patient.
  • the selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present disclosure employed or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated and like factors well known in the medical arts.
  • a “therapeutically effective dosage” of an anti-CD70 antibody preferably results in a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods or a prevention of impairment or disability due to the disease affliction.
  • a "therapeutically effective dosage” preferably inhibits cell growth or tumor growth by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 60% and still more preferably by at least about 80% relative to untreated subjects.
  • the ability of a compound to inhibit tumor growth can be evaluated in an animal model system predictive of efficacy in human tumors.
  • this property of a composition can be evaluated by examining the ability of the compound to inhibit, such inhibition in vitro by assays known to the skilled practitioner.
  • a therapeutically effective amount of a therapeutic compound can decrease tumor size or otherwise ameliorate symptoms in a subject.
  • One of ordinary skill in the art would be able to determine such amounts based on such factors as the subject's size, the severity of the subject's symptoms and the particular composition or route of administration selected.
  • a composition can be administered via one or more routes of administration using one or more of a variety of methods known in the art.
  • routes and/or mode of administration will vary depending upon the desired results.
  • Preferred routes of administration for antibodies of the disclosure include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, for example by injection or infusion.
  • parenteral administration means modes of administration other than enteral and topical administration, usually by injection and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.
  • an antibody can be administered via a non-parenteral route, such as a topical, epidermal or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually or topically.
  • a non-parenteral route such as a topical, epidermal or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually or topically.
  • the active compounds can be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches and microencapsulated delivery systems.
  • a controlled release formulation including implants, transdermal patches and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978 .
  • compositions can be administered with medical devices known in the art.
  • a therapeutic composition of the disclosure can be administered with a needleless hypodermic injection device, such as the devices disclosed in U.S. Patent Nos. 5,399,163 ; 5,383,851 ; 5,312,335 ; 5,064,413 ; 4,941,880 ; 4,790,824 ; or 4,596,556 .
  • a needleless hypodermic injection device such as the devices disclosed in U.S. Patent Nos. 5,399,163 ; 5,383,851 ; 5,312,335 ; 5,064,413 ; 4,941,880 ; 4,790,824 ; or 4,596,556 .
  • Examples of well-known implants and modules useful in the present disclosure include: U.S. Patent No, 4,487,603 , which discloses an implantable micro-infusion pump for dispensing medication at a controlled rate; U.S. Patent No.
  • U.S. Patent No. 4,447,233 which discloses a medication infusion pump for delivering medication at a precise infusion rate
  • U.S. Patent No. 4,447,224 which discloses a variable flow implantable infusion apparatus for continuous drug delivery
  • U.S. Patent No. 4,439,196 which discloses an osmotic drug delivery system having multi-chamber compartments
  • U.S. Patent No. 4,475,196 which discloses an osmotic drug delivery system.
  • Many other such implants, delivery systems and modules are known to those skilled in the art.
  • Human monoclonal antibodies can be formulated to ensure proper distribution in vivo.
  • the blood-brain barrier excludes many highly hydrophilic compounds.
  • therapeutic compounds cross the BBB (if desired)
  • they can be formulated, for example, in liposomes.
  • liposomes For methods of manufacturing liposomes, see, e.g., U.S. Patents 4,522,811 ; 5,374,548 ; and 5,399,331 .
  • the liposomes may comprise one or more moieties which are selectively transported into specific cells or organs, thus enhance targeted drug delivery (see, e.g., V. V. Ranade (1989) J Clin. Pharmacol. 29:685 ).
  • Exemplary targeting moieties include folate or biotin (see, e.g., U.S. Patent 5,416,016 to Low et al ); mannosides ( Umezawa et al, (1988) Biochem. Biophys. Res. Commun. 153:1038 ); antibodies ( P.G. Bloeman et al. (1995) FEBS Lett. 357:140 ; M. Owais et al. (1995) Antimicrob. Agents Chemother. 39:180 ); surfactant protein A receptor ( Briscoe et al (1995) Am. J. Physiol. 1233:134 ); p120 ( Schreier et al. (1994) J. Biol. Chem. 269:9090 ); see also K. Keinanen; M.L. Laukkanen (1994) FEBS Lett. 346:123 ; JJ. Killion; IJ. Fidler (1994; Immunomethods 4:273 .
  • the antibodies particularly the human antibodies, antibody compositions and methods disclosed herein have numerous in vitro and in vivo diagnostic and therapeutic utilities involving the diagnosis and treatment of CD70 mediated disorders.
  • these molecules can be administered to cells in culture, in vitro or ex vivo or to human subjects, e.g., in vivo, to treat, prevent and to diagnose a variety of disorders.
  • the term "subject” is intended to include human and non-human animals.
  • the term "non-human animals” includes all vertebrates, e.g ., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians and reptiles.
  • Preferred subjects include human patients having disorders mediated by CD70 activity.
  • the methods are particularly suitable for treating human patients having a disorder associated with aberrant CD70 expression.
  • the two can be administered in either order or simultaneously.
  • the antibodies can be used to specifically detect CD70 expression on the surface of cells and, moreover, can be used to purify CD70 via immunoaffmity purification.
  • CD70 is expressed in a variety of human cancers, including renal cell carcinomas, metastatic breast cancers, brain tumors, leukemias, lymphomas and nasopharangeal carcinomas ( Junker et al. (2005) J Urol. 173:2150-3 ; Sloan et al. (2004) Am J Pathol. 164:315-23 ; Held-Feindt and Mentlein (2002) Int J Cancer 98:352-6 ; Hishima et al. (2000) Am J Surg Pathol. 24: 742-6 ; Lens et al. (1999) Br J Haematol. 106:491-503 ).
  • An anti-CD70 antibody may be used alone to inhibit the growth of cancerous tumors. Alternatively, an anti-CD70 antibody may be used in conjunction with other immunogenic agents, standard cancer treatments or other antibodies, as described below.
  • Preferred cancers whose growth may be inhibited using the antibodies of the invention include cancers typically responsive to immunotherapy.
  • preferred cancers for treatment include renal cancer (e.g ., renal cell carcinoma), breast cancer, brain tumors, chronic or acute leukemias including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, lymphomas (e.g ., Hodgkin's and non-Hodgkin's lymphoma, lymphocytic lymphoma, primary CNS lymphoma, T-cell lymphoma) and nasopharangeal carcinomas.
  • renal cancer e.g ., renal cell carcinoma
  • breast cancer e.g ., brain tumors
  • chronic or acute leukemias including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, lymphomas (e.g ., Hodgkin'
  • cancers examples include melanoma (e.g. , metastatic malignant melanoma), prostate cancer, colon cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, solid tumors of childhood, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of
  • the human antibodies, antibody compositions and methods of the invention can be used to treat a subject with a tumorigenic disorder, e.g ., a disorder characterized by the presence of tumor cells expressing CD70 including, for example, renal cell carcinomas (RCC), such as clear cell RCC, glioblastoma, breast cancer, brain tumors, nasopharangeal carcinomas, non-Hodgkin's lymphoma (NHL), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), Burkitt's lymphoma, anaplastic large-cell lymphomas (ALCL), multiple myeloma, cutaneous T-cell lymphomas, nodular small cleaved-cell lymphomas, lymphocytic lymphomas, peripheral T-cell lymphomas, Lennert's lymphomas, immunoblastic lymphomas, T-cell leukemia/lymphomas (ATLL), adult
  • RCC renal cell carcinomas
  • NHL non
  • an anti-CD70 antibody or antigen-binding portion thereof comprising administering to the subject a therapeutically effective amount of an anti-CD70 antibody or antigen-binding portion thereof.
  • the antibody is a human anti-CD70 antibody (such as any of the human anti-human CD70 antibodies of the invention).
  • the antibodies can detect levels of CD70 or levels of cells which contain CD70 on their membrane surface, which levels can then be linked to certain disease symptoms.
  • the antibodies can be used to inhibit or block CD70 function which, in turn, can be linked to the prevention or amelioration of certain disease symptoms, thereby implicating CD70 as a mediator of the disease. This can be achieved by contacting an experimental sample and a control sample with the anti-CD70 antibody under conditions that allow for the formation of a complex between the antibody and CD70. Any complexes formed between the antibody and CD70 are detected and compared in the experimental sample and the control.
  • compositions can be tested using the flow cytometric assays described in the Examples below.
  • the antibodies, immunoconjugates and compositions of the invention have additional utility in therapy and diagnosis of CD70-related diseases.
  • they can be used to elicit in vivo or in vitro one or more of the following biological activities: to inhibit the growth of and/or kill a cell expressing CD70; to mediate phagocytosis or ADCC of a cell expressing CD70 in the presence of human effector cells; or to block CD70 ligand binding to CD70.
  • CD70-related diseases include cancer, renal cell carcinomas (RCC), such as clear cell RCC, glioblastoma, breast cancer, brain tumors, nasopharangeal carcinomas, non-Hodgkin's lymphoma, acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), Burkitt's lymphoma, anaplastic large-cell lymphomas (ALCL), multiple myeloma, cutaneous T-cell lymphomas, nodular small cleaved-cell lymphomas, lymphocytic lymphomas, peripheral T-cell lymphomas, Lennert's lymphomas, immunoblastic lymphomas, T-cell leukemia/lymphomas (ATLL), adult T-cell leukemia (T-ALL), entroblastic/centrocytic (cb
  • Suitable routes of administering antibody compositions in vivo and in vitro are well known in the art and can be selected by those of ordinary skill.
  • the antibody compositions can be administered by injection ( e.g ., intravenous or subcutaneous).
  • Suitable dosages of the molecules used will depend on the age and weight of the subject and the concentration and/or formulation of the antibody composition.
  • human anti-CD70 antibodies can be coadministered with one or other more therapeutic agents, e.g. , a cytotoxic agent, a radiotoxic agent or an immunosuppressive agent.
  • the antibody can be linked to the agent (as an immunocomplex) or can be administered separate from the agent. In the latter case (separate administration), the antibody can be administered before, after or concurrently with the agent or can be coadministered with other known therapies, e.g., an anti-cancer therapy, e.g., radiation.
  • Such therapeutic agents include, among others, anti-neoplastic agents such as doxorubicin (adriamycin), cisplatin bleomycin sulfate, carmustine, chlorambucil and cyclophosphamide hydroxyurea which, by themselves, are only effective at levels which are toxic or subtoxic to a patient.
  • Cisplatin is intravenously administered as a 100 mg/ dose once every four weeks and adriamycin is intravenously administered as a 60-75 mg/ml dose once every 21 days.
  • Co-administration of human anti-CD70 antibodies or antigen binding fragments thereof with chemotherapeutic agents provides two anticancer agents which operate via different mechanisms which yield a cytotoxic effect to human tumor cells. Such co-administration can solve problems due to development of resistance to drugs or a change in the antigenicity of the tumor cells which would render them unreactive with the antibody.
  • Target-specific effector cells e.g., effector cells linked to compositions (e.g., human antibodies, multispecific and bispecific molecules) can also be used as therapeutic agents.
  • Effector cells for targeting can be human leukocytes such as macrophages, neutrophils or monocytes. Other cells include eosinophils, natural killer cells and other IgG- or IgA-receptor bearing cells. If desired, effector cells can be obtained from the subject to be treated.
  • the target-specific effector cells can be administered as a suspension of cells in a physiologically acceptable solution.
  • the number of cells administered can be in the order of 10 8 -10 9 but will vary depending on the therapeutic purpose. In general, the amount will be sufficient to obtain localization at the target cell, e.g., a tumor cell expressing CD70 and to effect cell killing by, e.g ., phagocytosis. Routes of administration can also vary.
  • Target-specific effector cells can be performed in conjunction with other techniques for removal of targeted cells.
  • anti-tumor therapy using compositions e.g ., human antibodies, multispecific and bispecific molecules
  • effector cells armed with these compositions can be used in conjunction with chemotherapy.
  • combination immunotherapy may be used to direct two distinct cytotoxic effector populations toward tumor cell rejection.
  • anti-CD70 antibodies linked to anti-Fc-gamma RI or anti-CD3 may be used in conjunction with IgG- or IgA-receptor specific binding agents.
  • compositions which have complement binding sites, such as portions from IgG1, -2 or -3 or IgM which bind complement, can also be used in the presence of complement.
  • complement binding sites such as portions from IgG1, -2 or -3 or IgM which bind complement
  • Ex vivo treatment of a population of cells comprising target cells with a binding agent as disclosed herein and appropriate effector cells can be supplemented by the addition of complement or serum containing complement.
  • Phagocytosis of target cells coated with a binding agent of the disclosure can be improved by binding of complement proteins.
  • Target cells coated with the compositions e.g ., human antibodies
  • the compositions may not activate complement.
  • compositions e .g., human antibodies and immunoconjugates
  • compositions may comprise human antibodies and serum or complement. These compositions are advantageous in that the complement is located in close proximity to the human antibodies. Alternatively, the human antibodies and the complement or serum can be administered separately.
  • kits comprising the antibody compositions of the invention (e.g ., human antibodies or immunoconjugates) and instructions for use.
  • the kit can further contain one ore more additional reagents, such as an immunosuppressive reagent, a cytotoxic agent or a radiotoxic agent or one or more additional human antibodies (e.g., a human antibody having a complementary activity which binds to an epitope in the CD70 antigen distinct from the first human antibody).
  • patients treated with antibody compositions of the invention can be additionally administered (prior to, simultaneously with or following administration of a human antibody of the invention) with another therapeutic agent, such as a cytotoxic or radiotoxic agent, which enhances or augments the therapeutic effect of the human antibodies.
  • another therapeutic agent such as a cytotoxic or radiotoxic agent, which enhances or augments the therapeutic effect of the human antibodies.
  • the subject can be additionally treated with an agent that modulates, e.g ., enhances or inhibits, the expression or activity of Fc ⁇ or Fc ⁇ receptors by, for example, treating the subject with a cytokine.
  • an agent that modulates e.g ., enhances or inhibits, the expression or activity of Fc ⁇ or Fc ⁇ receptors by, for example, treating the subject with a cytokine.
  • Preferred cytokines for administration during treatment with the multispecific molecule include of granulocyte colony-stimulating factor (G-CSF), granulocyte- macrophage colony-stimulating factor (GM-CSF), interferony (IFN- ⁇ ) and tumor necrosis factor (TNF).
  • G-CSF granulocyte colony-stimulating factor
  • GM-CSF granulocyte- macrophage colony-stimulating factor
  • IFN- ⁇ interferony
  • TNF tumor necrosis factor
  • compositions (e.g ., human antibodies) of the invention can also be used to target cells expressing Fc ⁇ R or CD70, for example for labeling such cells.
  • the binding agent can be linked to a molecule that can be detected.
  • the detectable label can be, e.g., a radioisotope, a fluorescent compound, an enzyme or an enzyme co-factor.
  • an anti-CD70 antibody can be conjugated to any of the toxin compounds described in US Patent Nos. 6,281,354 and 6,548,530 , US patent publication Nos. 20030050331 , 20030064984 , 20030073852 and 20040087497 or published in WO 03/022806 .
  • the disclosure also provides methods for localizing ex vivo or in vivo cells expressing CD70 (e.g., with a detectable label, such as a radioisotope, a fluorescent compound, an enzyme or an enzyme co-factor).
  • a detectable label such as a radioisotope, a fluorescent compound, an enzyme or an enzyme co-factor.
  • the immunoconjugates can be used to kill cells which have CD70 cell surface receptors by targeting cytotoxins or radiotoxins to CD70.
  • Immunization protocols utilized as antigen recombinant human CD70 fused with a dual myc-His tag were used in some immunizations.
  • Fully human monoclonal antibodies to CD70 were prepared using the HCo7, HCo12 and HCo17 strains of HuMab transgenic mice and the KM strain of transgenic transchromosomic mice, each of which express human antibody genes.
  • the endogenous mouse kappa light chain gene has been homozygously disrupted as described in Chen et al. (1993) EMBO J. 12:811-820 and the endogenous mouse heavy chain gene has been homozygously disrupted as described in Example 1 of PCT Publication WO 01/09187 .
  • this mouse strain carries a human kappa light chain transgene, KCo5, as described in Fishwild et al.
  • the KM Mouse ® strain contains the SC20 transchromosome as described in PCT Publication WO 02/43478 .
  • mice of the HuMAb Mouse ® and KM Mouse ® were immunized with recombinant human CD70 as antigen or whole cells expressing CD70 on the cell surface.
  • General immunization schemes for HuMab mice are described in Lonberg, N. et al (1994) Nature 368(6474): 856-859 ; Fishwild, D. et al. (1996) Nature Biotechnology 14: 845-851 and PCT Publication WO 98/24884 .
  • the mice were 6-16 weeks of age upon the first infusion of antigen. 5-10x10 6 cells were used to immunize the HuMab mice intraperitonealy (IP), subcutaneously (Sc) or via footpad injection.
  • IP intraperitonealy
  • Sc subcutaneously
  • mice were immunized twice with antigen in complete Freund's adjuvant or Ribi adjuvant IP, followed by 3-21 days IP (up to a total of 11 immunizations) with the antigen in incomplete Freund's or Ribi adjuvant.
  • the immune response was monitored by retroorbital bleeds.
  • the plasma was screened by ELISA and FACS (as described below) and mice with sufficient titers of anti-CD70 human immunogolobulin were used for fusions.
  • Mice were boosted intravenously with antigen 3 days before sacrifice and removal of the spleen. Typically, 10-35 fusions for each antigen were performed. Several dozen mice were immunized for each antigen.
  • Flow cytometric analyses were performed using a FACSCalibur flow cytometry (Becton Dickinson, San Jose, CA). Antibodies that bound to the CD70 expressing CHO cells but not the non-CD70 expressing parental CHO cells were further tested for binding to CD70 by ELISA, as described by Fishwild, D. et al. (1996). Briefly, microtiter plates were coated with purified recombinant CD70 fusion protein from transfected CHO cells at 1-2 ⁇ g /ml in PBS, 100 ⁇ l/wells incubated 4 °C overnight then blocked with 200 ⁇ l/well of 5% chicken serum in PBS/Tween (0.05%).
  • mice splenocytes isolated from a HuMab mouse ® and/or a KM mouse ® , were fused to a mouse myeloma cell line either using PEG based upon standard protocols or electric field based electrofusion using a Cyto Pulse large chamber cell fusion electroporator (Cyto Pulse Sciences, Inc., Glen Burnie, MD). The resulting hybridomas were then screened for the production of antigen-specific antibodies. Single cell suspensions of splenocytes from immunized mice were fused to one-fourth the number of SP2/0 nonsecreting mouse myeloma cells (ATCC, CRL 1581) with 50% PEG (Sigma).
  • DMEM high glucose medium with L-glutamine and sodium pyruvate (Mediatech, Inc., Herndon, VA) and further containing 10% fetal Bovine Serum (Hyclone, Logan, UT), 18% P388DI conditional media, 5% Origen Hybridoma cloning factor (BioVeris, Gaithersburg, VA), 4 mM L-glutamine, 5mM HEPES, 0.055 mM ⁇ -mercaptoethanol, 50 units/ml penicillin, 50 mg/ml streptomycin and 1X Hypoxanthine-aminopterin-thymidine (HAT) media (Sigma; the HAT is added 24 hours after the fusion).
  • HAT Hypoxanthine-aminopterin-thymidine
  • Hybridoma clones 2H5, 10B4, 8B5, 18E7 and 69A7 were selected for further analysis.
  • the cDNA sequences encoding the heavy and light chain variable regions of the 2H5, 10B4, 8B5, 18E7 and 69A7 monoclonal antibodies were obtained from the 2H5, 10B4, 8B5, 18E7 and 69A7 hybridomas, respectively, using standard PCR techniques and were sequenced using standard DNA sequencing techniques.
  • nucleotide and amino acid sequences of the heavy chain variable region of 2H5 are shown in Figure 1A and in SEQ ID NO:41 and 1, respectively.
  • nucleotide and amino acid sequences of the light chain variable region of 2H5 are shown in Figure 1B and in SEQ ID NO:46 and 6, respectively.
  • nucleotide and amino acid sequences of the heavy chain variable region of 10B4 are shown in Figure 2A and in SEQ ID NO:42 and 2, respectively.
  • nucleotide and amino acid sequences of the light chain variable region of 10B4 are shown in Figure 2B and in SEQ ID NO:47 and 7, respectively.
  • nucleotide and amino acid sequences of the heavy chain variable region of 8B5 are shown in Figure 3A and in SEQ ID NO:43 and 3, respectively.
  • nucleotide and amino acid sequences of the light chain variable region of 8B5 are shown in Figure 3B and in SEQ ID NO:48 and 8, respectively.
  • nucleotide and amino acid sequences of the heavy chain variable region of 18E7 are shown in Figure 4A and in SEQ ID NO:44 and 4, respectively.
  • nucleotide and amino acid sequences of the light chain variable region of 18E7 are shown in Figure 4B and in SEQ ID NO:49 and 9, respectively.
  • nucleotide and amino acid sequences of the heavy chain variable region of 69A7 are shown in Figure 5A and in SEQ ID NO:45 and 5, respectively.
  • nucleotide and amino acid sequences of the light chain variable region of 69A7 are shown in Figure 5B and in SEQ ID NO:50 and 10, respectively.
  • a comparison of anti-CD70 antibodies on binding to immunopurified CD70 was performed by standard ELISA to examine the specificity of binding for CD70.
  • Recombinant myc-tagged CD70 was coated on a plate overnight., then tested for binding against the anti-CD70 human monoclonal antibodies 2H5, 10B4, 8B5, 18E7 and 69A7. Standard ELISA procedures were performed.
  • the anti-CD70 human monoclonal antibodies were added at a concentration of 1 ⁇ g/ml and titrated down at 1:2 serial dilutions.
  • Goat-anti-human IgG (Fc or kappa chain-specific) polyclonal antibody conjugated with horseradish peroxidase (HRP) was used as secondary antibody. The results are shown in Figure 13 .
  • the anti-CD70 human monoclonal antibodies 2H5, 10B4, 8B5 and 18E7 bound with high specificity to CD70.
  • Anti-CD70 antibodies were tested for binding to renal cell carcinoma cells expressing CD70 on their cell surface by flow cytometry.
  • the renal cell carcinoma cell lines A-498 (ATCC Accession No. HTB-44), 786-O (ATCC Accession No. CRL-1932), ACHN (ATCC Accession No. CRL-1611), Caki-1 (ATCC Accession No. HTB-46) and Caki-2 (ATCC Accession No. HTB-47) were each tested for antibody binding. Binding of the HuMAb 2H5 anti-CD70 human monoclonal antibody was assessed by incubating 1x10 5 cells with 2H5 at a concentration of 1 ⁇ g/ml. The cells were washed and binding was detected with a FITC-labeled anti-human IgG Ab.
  • the renal cell carcinoma cell lines 786-O and A-498 were tested for binding of the HuMAb anti-CD70 human monoclonal antibodies 2H5, 8B5, 10B4 and 18E7 at different concentrations. Binding of the anti-CD70 human monoclonal antibodies was assessed by incubating 5x10 5 cells with antibody at a starting concentration of 50 ⁇ g/ml and serially diluting the antibody at a 1:3 dilution. The cells were washed and binding was detected with a PE-labeled anti-human IgG Ab. Flow cytometric analyses were performed using a FACSCalibur flow cytometry (Becton Dickinson, San Jose, CA).
  • FIG. 15A (786-O) and Figure 15B (A-498).
  • the anti-CD70 monoclonal antibodies 2H5, 8B5, 10B4 and 18E7 bound to the renal carcinoma cell lines 786-O and A-498 in a concentration dependent manner, as measured by the mean fluorescent intensity (MFI) of staining.
  • MFI mean fluorescent intensity
  • the EC 50 values for the anti-CD70 monoclonal antibodies ranged from 1.844 nM to 6.669 nM for the 786-O cell line and 3.984 nM to 11.84 nM for the A-498 cell line.
  • Binding of the HuMAb 2H5 and 69A7 anti-CD70 human monoclonal antibodies to the renal cell carcinoma cell line 786-O was assessed by incubating 2x10 5 cells with either 2H5 or 69A7 at a concentration of 10 ⁇ g/ml. An isotype control antibody was used as a negative control. The cells were washed and binding was detected with a FITC-labeled anti-human IgG Ab. Flow cytometric analyses were performed using a FACSCalibur flow cytometry (Becton Dickinson, San Jose, CA). The results are shown in Figure 15C . Both anti-CD70 monoclonal antibodies bound to the renal carcinoma cell line 786-O.
  • the renal cell carcinoma cell line 786-O was tested for binding of the HuMAb anti-CD70 human monoclonal antibody 69A7 at different concentrations. Binding of the anti-CD70 human monoclonal antibodies was assessed by incubating 5x10 5 cells with antibody at a starting concentration of 10 ⁇ g/ml and serially diluting the antibody at a 1:3 dilution. The cells were washed and binding was detected with a PE-labeled anti-human IgG Ab. Flow cytometric analyses were performed using a FACSCalibur flow cytometry (Becton Dickinson, San Jose, CA). The results are shown in Figure 15D .
  • the anti-CD70 monoclonal antibody 69A7 bound to the renal carcinoma cell line 786-O in a concentration dependent manner, as measured by the mean fluorescent intensity (MFI) of staining.
  • MFI mean fluorescent intensity
  • Anti-CD70 antibodies were tested for binding to lymphoma cells expressing CD70 on their cell surface by flow cytometry.
  • the lymphoma cell lines Daudi (ATCC Accession No. CCL-213), HuT 78 (ATCC Accession No. TIB-161) and Raji (ATCC Accession No. CCL-86) were each tested for antibody binding. Binding of the HuMAb 2H5 anti-CD70 human monoclonal antibody was assessed by incubating 1x10 5 cells with 2H5 at a concentration of 1 ⁇ g/ml. The cells were washed and binding was detected with a FITC-labeled anti-human IgG Ab. The Jurkat cell line, which does not express CD70 on the cell surface, was used as a negative control.
  • Flow cytometric analyses were performed using a FACSCalibur flow cytometry (Becton Dickinson, San Jose, CA). The results are shown in Figure 16 .
  • the anti-CD70 monoclonal antibody 2H5 bound to the lymphoma cell lines Daudi, HuT 78 and Raji, as measured by the mean fluorescent intensity (MFI) of staining.
  • the lymphoma cell lines Raji and Granta 519 were tested for binding of the HuMAb anti-CD70 human monoclonal antibody 2H5 at Varying concentrations. Binding of the anti-CD70 human monoclonal antibodies was assessed by incubating 5x10 5 cells with antibody at a starting concentration of 50 ⁇ g/ml and serially diluting the antibody at a 1:3 dilution. An isotype control antibody was used as a negative control. The cells were washed and binding was detected with a PE-labeled anti-human IgG Ab. Flow cytometric analyses were performed using a FACSCalibur flow cytometry (Becton Dickinson, San Jose, CA).
  • Binding of the HuMAbs 2H5 and 69A7 anti-CD70 human monoclonal antibodies to the Raji lymphoma cell line was assessed by incubating 2x10 5 cells with HuMAb at a concentration of 10 ⁇ g/ml. The cells were washed and binding was detected with a FITC-labeled anti-human IgG Ab. An isotype control antibody and secondary antibody alone were used as negative control. Flow cytometric analyses were performed using a FACSCalibur flow cytometry (Becton Dickinson, San Jose, CA). The results are shown in Figure 17C . Both anti-CD70 monoclonal antibodies bound to the Raji lymphoma cell line, as measured by the mean fluorescent intensity (MFI) of staining.
  • MFI mean fluorescent intensity
  • a competition FACS assay was carried out to elucidate the binding specificity of 69A7 against 2H5.
  • Raji cells were incubated with either naked 69A7, 2H5, an isotype control antibody or no antibody at a concentration of 10 ⁇ g/ml. After wash, the cells were incubated with FITC-conjugated 69A7 at a concentration of 10 ⁇ g/ml. The cells were washed and binding was detected with a FITC-labeled anti-human IgG Ab.
  • Flow cytometric analyses were performed using a FACSCalibur flow cytometry (Becton Dickinson, San Jose, CA). The results are shown in Figure 17D . Both the anti-CD70 antibody 69A7 and 2H5 blocked binding of FITC-labelled 69A7, indicating that both 2H5 and 69A7 share a similar binding epitope.
  • the Daudi lymphoma cell line and 786-O renal carcinoma cell were further tested for antibody binding. Binding of the HuMAb 69A7 anti-CD70 human monoclonal antibody was assessed by incubating 2x10 5 cells with 69A7 at a concentration of 1 ⁇ g/ml. The cells were washed and binding was detected with a FITC-labeled anti-human IgG Ab. The Jurkat cell line, which does not express CD70 on the cell surface, was used as a negative control. Flow cytometric analyses were performed using a FACSCalibur flow cytometry (Becton Dickinson, San Jose, CA). The results are shown in Figure 17E . The anti-CD70 monoclonal antibody 69A7 bound to the Daudi lymphoma cell line and 786-O renal carcinoma cell line, as measured by the mean fluorescent intensity (MFI) of staining.
  • MFI mean fluorescent intensity
  • the binding affinity of the 2H5, 8B5, 10B4 and 18E7 monoclonal antibodies was tested for binding affinity to a CD70 transfected CHO cell line using a Scatchard analysis.
  • CHO cells were transfected with full length CD70 using standard techniques and grown in RPMI media containing 10% fetal bovine serum (FBS). The cells were trypsinized and washed once in Tris based binding buffer (24mM Tris pH 7.2, 137mM NaCl, 2.7mM KCl, 2mM Glucose, 1mM CaCl 2 , 1mM MgCl 2 , 0.1% BSA) and the cells were adjusted to 2x10 6 cells/ml in binding buffer. Millipore plates (MAFB NOB) were coated with 1% nonfat dry milk in water and stored a 4 °C overnight. The plates were washed three times with 0.2ml of binding buffer.
  • Tris based binding buffer 24mM Tris pH 7.2, 137mM NaCl, 2.7mM KCl, 2mM Glucose, 1mM CaCl 2 , 1mM MgCl 2 , 0.1% BSA
  • Millipore plates MAFB NO
  • Millipore plates were washed three times with 0.2 ml of cold wash buffer (24mM Tris pH 7.2, 500mM NaCl, 2.7mM KCl, 2mM Glucose, 1mM CaCl 2 , 1mM MgCl 2 , 0.1% BSA.). The filters were removed and counted in a gamma counter. Evaluation of equilibrium binding was performed using single site binding parameters with the Prism software (San Diego, CA).
  • the K D of the antibody for CD70 transfected CHO cells was approximately 2.1nM for 2H5, 5.1nM or 8B5, 1.6nM for 10B4 and 1.5nM for 18E7.
  • Anti-CD70 HuMAbs were tested for the ability to internalize into CD70-expressing renal carcinoma cells using a Hum-Zap internalization assay.
  • the Hum-Zap assay tests for internalization of a primary human antibody through binding of a secondary antibody with affinity for human IgG conjugated to the toxin saporin.
  • the CD70-expressing renal carcinoma cancer cell line 786-O was seeded at 1.25x10 4 cells/well in 100 ⁇ l wells overnight.
  • the anti-CD70 HuMAb antibodies 2H5, 8B5, 10B4 or 18E7 were added to the wells at a starting concentration of 30 nM and titrated down at 1:3 serial dilutions.
  • An isotype control antibody that is non-specific for CD70 was used as a negative control.
  • the Hum-Zap Advanced Targeting Systems, San Diego, CA, IT-22-25 was added at a concentration of 11 nM and plates were allowed to incubate for 72 hours.
  • the plates were then pulsed with 1.0 ⁇ Ci of 3 H-thymidine for 24 hours, harvested and read in a Top Count Scintillation Counter (Packard Instruments, Meriden, CT).
  • the results are shown in Figure 18 .
  • the anti-CD70 antibodies 2H5, 8B5, 10B4 and 18E7 showed an antibody concentration dependent decrease in 3 H-thymidine incorporation in CD70-expressing 786-O renal carcinoma cancer cells.
  • the EC 50 value for the anti-CD70 antibody 2H5 was 0.9424 nM. This data demonstrates that the anti-CD70 antibodies 2H5, 8B5, 10B4 and 18E7 internalize into a renal carcinoma cancer cell line.
  • Example 8 Assessment of cell killing of a toxin-conjugated anti-CD70 antibody on renal cell carcinoma cell lines
  • anti-CD70 monoclonal antibodies conjugated to a toxin were tested for the ability to kill CD70+ renal cell carcinoma cell lines in a cell proliferation assay.
  • the anti-CD70 HuMAb antibodies 2H5, 8B5, 10B4 or 18E7 were conjugated to a toxin via a linker, such as a peptidyl, hydrazone or disulfide linker.
  • the CD70-expressing renal carcinoma cancer cell lines ACHN and Caki-2 were seeded at 2.5x10 4 cells/wells and the CD70-expressing renal carcinoma cancer cell line 786-O was seeded at 1.25x10 4 cells/wells in 100 ⁇ l wells for 3 hours.
  • An anti-CD70 antibody-toxin conjugate was added to the wells at a starting concentration of 30 nM and titrated down at 1:3 serial dilutions.
  • An isotype control antibody that is non-specific for CD70 was used as a negative control. Plates were allowed to incubate for 69 hours. The plates were then pulsed with 1.0 ⁇ Ci of 3 H-thymidine for 24 hours, harvested and read in a Top Count Scintillation Counter (Packard Instruments, Meriden, CT). The results are shown in Figures 19A (Caki-2), 19B (786-O) and 19C (ACHN). The anti-CD70 antibodies 2H5, 8B5, 10B4 and 18E7 showed an antibody-toxin concentration dependent decrease in 3 H-thymidine incorporation in CD70-expressing Caki-2, 786-O and ACHN renal carcinoma cancer cells.
  • the EC 50 values for the anti-CD70 antibodies ranged from 6.728 nM to 76.05 nM in the CAKI-2 cells, 1.635 nM to 3.940 nM in the 786-O cells and 9.406 nM to 108.5 nM in the ACHN cells.
  • This data demonstrates that the anti-CD70 antibodies 2H5, 8B5, 10B4 and 18E7 are cytotoxic to renal carcinoma cancer cells when conjugated to a toxin.
  • anti-CD70 monoclonal antibodies were tested for the ability to kill CD70+ cell lines in the presence of effector cells via antibody dependent cellular cytotoxicity (ADCC) in a fluorescence cytotoxicity assay.
  • ADCC antibody dependent cellular cytotoxicity
  • Human effector cells were prepared from whole blood as follows. Human peripheral blood mononuclear cells were purified from heparinized whole blood by standard Ficoll-paque separation. The cells were resuspended in RPMI1640 media containing 10% FBS and 200 U/ml of human IL-2 and incubated overnight at 37°C. The following day, the cells were collected and washed four times in culture media and resuspended at 2 x 10 7 cells/ml. Target CD70+ cells were incubated with BATDA reagent (Perkin Elmer, Wellesley, MA) at 2.5 ⁇ l BATDA per 1 x 10 6 target cells/mL for 20 minutes at 37° C. The target cells were washed four times, spun down and brought to a final volume of 1x10 5 cells/ml.
  • BATDA reagent Perkin Elmer, Wellesley, MA
  • the CD70+ cell lines ARH-77 human B lymphoblast leukemia; ATCC Accession No. CRL-1621
  • HuT 78 human cutaneous lymphocyte lymphoma; ATCC Accession No. TIB-161
  • Raji human B lymphocyte Burkitt's lymphoma; ATCC Accession No. CCL-86
  • a negative control cell line L540 human Hodgkin's lymphoma; DSMZ Deposit No. ACC 72
  • a target to effector ratio of 1:50 was used throughout the experiments.
  • a human IgG1 isotype control was used as a negative control.
  • the supernatants were collected, quick spun again and 20 ⁇ l of supernatant was transferred to a flat bottom plate, to which 180 ⁇ l of Eu solution (Perkin Elmer, Wellesley, MA) was added and read in a RubyStar reader (BMG Labtech).
  • the % lysis was calculated as follows: (sample release - spontaneous release * 100) / (maximum release - spontaneous release), where the spontaneous release is the fluorescence from wells which only contain target cells and maximum release is the fluorescence from wells containing target cells and have been treated with 2% Triton-X. Cell cytotoxicity % lysis for the ARH-77, HuT 78, Raji and L-540 cell lines are shown in Figures 20A-D , respectively.
  • Example 10 Assessment of cell killing of a toxin-conjugated anti-CD70 antibody on human lymphoma cell lines
  • anti-CD70 monoclonal antibodies conjugated to a toxin were tested for the ability to kill CD70+ human lymphoma cell lines in a cell proliferation assay.
  • the anti-CD70 HuMAb antibody 2H5 was conjugated to a toxin via a linker, such as a peptidyl, hydrazone or disulfide linker.
  • a linker such as a peptidyl, hydrazone or disulfide linker.
  • Examples of toxin compounds that may be conjugated to the antibodies of the current disclosure are described in the concurrently filed application with Attorney Docket No. 04280/100M629US3, filed on September 26, 2005.
  • the CD70-expressing human lymphoma cancer cell lines Daudi, HuT 78, Granta 519 and Raji were seeded at 10 5 cells/well in 100 ⁇ l wells for 3 hours.
  • An anti-CD70 antibody-toxin conjugate was added to the wells at a starting concentration of 30 nM and titrated down at 1:2 serial dilutions.
  • the HuMAb antibody 2H5-toxin conjugate was also tested on Jurkat cells, a negative control cell line that does not express CD70 on the cell surface. Plates were allowed to incubate for 72 hours. The plates were then pulsed with 0.5 ⁇ Ci of 3 H-thymidine for 8 hours before termination of the culture, harvested and read in a Top Count Scintillation Counter (Packard Instruments). Figure 21 showed the effects of the 2H5-conjugate on the Daudi, HuT 78, Granta 519 and Jurkat cells.
  • the anti-CD70 antibody 2H5 showed an antibody-toxin concentration dependent decrease in 3 H-thymidine incorporation in CD70-expressing Daudi, HuT 78 and Granta 519 B-cell lymphoma cancer cells, but not in the Jurkat cells.
  • the CD70-expressing human lymphoma cancer cell line Raji was seeded at 10 4 cells/well in 100 ⁇ l wells for 3 hours.
  • An anti-CD70 antibody-toxin conjugate was added to the wells at a starting concentration of 30 nM and titrated down at 1:3 serial dilutions.
  • a toxin-conjugate isotype control antibody was used as a control. Plates were allowed to incubate for 72 hours with either a wash at 3 hours or a continuous wash. The plates were then pulsed with 0.5 ⁇ Ci of 3 H-thymidine for 8 hours before termination of the culture, harvested and read in a Top Count Scintillation Counter (Packard Instruments).
  • Figures 22A and 22B showed an antibody-toxin concentration dependent decrease in 3 H-thymidine incorporation on Raji cells with a 3 hour wash or with a continuous wash, respectively.
  • Example 11 Treatment of in vivo tumor xenograft model using naked and cytotoxin-conjugated anti-CD70 antibodies
  • mice implanted with a renal cell carcinoma tumor were treated in vivo with toxin-conjugated anti-CD70 antibodies to examine the in vivo effect of the antibodies on tumor growth.
  • A-498 (ATCC Accession No. HTB-44) and ACHN (ATCC Accession No. CRL-1611) cells were expanded in vitro using standard laboratory procedures.
  • Male Ncr athymic nude mice (Taconic, Hudson, NY) between 6-8 weeks of age were implanted subcutaneously in the right flank with 7.5 x10 6 ACHN or A-498 cells in 0.2 ml of PBS/Matrigel (1:1) per mouse. Mice were weighed and measured for tumors three dimensionally using an electronic caliper twice weekly after implantation. Tumor volumes were calculated as height x width x length. Mice with ACHN tumors averaging 270 mm 3 or A498 tumors averaging 110 mm 3 were randomized into treatment groups.
  • mice were dosed intraperitoneally with PBS vehicle, toxin-conjugated isotype control antibody or toxin-conjugated anti-CD70 HuMAb 2H5 on Day 0.
  • Examples of toxin compounds that may be conjugated to the antibodies of the current disclosure are described in the concurrently filed application with reference number MEDX-0034US4.
  • the mice in the A-498 sample group were tested with three different toxin compounds. Mice were monitored for tumor growth for 60 days post dosing. Mice were euthanized when the tumors reached tumor end point (2000 mm 3 ). The results are shown in Figure 23A (A-498 tumors) and 23B (ACHN tumors).
  • the anti-CD70 antibody 2H5 conjugated to a toxin extended the mean time to reaching the tumor end point volume (2000 mm 3 ) and slowed tumor growth progression.
  • treatment with an anti-CD70 antibody-toxin conjugate has a direct in vivo inhibitory effect on tumor growth.
  • Antibodies with reduced amounts of fucosyl residues have been demonstrated to increase the ADCC ability of the antibody.
  • the 2H5 HuMAb has been produced that is lacking in fucosyl residues.
  • the CHO cell line Ms704-PF which lacks the fucosyltransferase gene, FUT 8 (Biowa, Inc., Princeton, NJ) was electroporated with a vector which expresses the heavy and light chains of antibody 2H5.
  • Drug-resistant clones were selected by growth in Ex-Cell 325-PF CHO media (JRH Biosciences, Lenexa, KS) with 6 mM L-glutamine and 500 ⁇ g/ml G418 (Invitrogen, Carlsbad, CA). Clones were screened for IgG expression by standard ELISA assay. Two separate clones were produced, B8A6 and B8C11, which had production rates ranging from 1.0 to 3.8 picograms per cell per day.
  • Example 14 Assessment of ADCC activity of defucosylated anti-CD70 antibody
  • a defucosylated and non-defucosylated anti-CD70 monoclonal antibody was tested for the ability to kill CD70+ cells in the presence of effector cells via antibody dependent cellular cytotoxicity (ADCC) in a fluorescence cytotoxicity assay.
  • ADCC antibody dependent cellular cytotoxicity
  • Human Anti-CD70 monoclonal antibody 2H5 was defucosylated as described above.
  • Human effector cells were prepared from whole blood as follows. Human peripheral blood mononuclear cells were purified from heparinized whole blood by standard Ficoll-paque separation. The cells were resuspended in RPMI1640 media containing 10% FBS (culture media) and 200 U/ml of human IL-2 and incubated overnight at 37°C. The following day, the cells were collected and washed once in culture media and resuspended at 2 x 10 7 cells/ml.
  • Target CD70+ cells were incubated with BATDA reagent (Perkin Elmer, Wellesley, MA) at 2.5 ⁇ l BATDA per 1 x 10 6 target cells/mL in culture media supplemented with 2.5mM probenecid (assay media) for 20 minutes at 37° C.
  • BATDA reagent Perkin Elmer, Wellesley, MA
  • the target cells were washed four times in PBS with 20mM HEPES and 2.5mM probenecid, spun down and brought to a final volume of 1 x 10 5 cells/ml in assay media.
  • the CD70+ cell lines ARH-77 (human B lymphoblast leukemia; ATCC Accession No. CRL-1621), MEC-1 (human chronic B cell leukemia; DSMZ Accession No. ACC 497), SU-DHL-6 (human B cell lymphoma, DSMZ Accession No. Acc572), IM-9 (human B lymphoblast; ATCC Accession No. CCL-159) and HuT 78 (human cutaneous lymphocyte lymphoma; ATCC Accession No. TIB-161), were tested for antibody specific ADCC to the defucosylated and non-defucosylated human anti-CD70 monoclonal antibody 2H5 using the Delfia fluorescence emission analysis as follows.
  • the target cell line ARH77 (100 ⁇ l of labeled target cells) was incubated with 50 ⁇ l of effector cells and 50 ⁇ l of either 2H5 or defucosylated 2H5 antibody. A target to effector ratio of 1:50 was used throughout the experiments. A human IgG1 isotype control was used as a negative control. Following a 2100 rpm pulse spin and one hour incubation at 37° C, the supernatants were collected, quick spun again and 20 ⁇ l of supernatant was transferred to a flat bottom plate, to which 180 ⁇ l of Eu solution (Perkin Elmer, Wellesley, MA) was added and read in a Fusion Alpha TRF plate reader (Perkin Elmer).
  • the % lysis was calculated as follows: (sample release - spontaneous release * 100) / (maximum release - spontaneous release), where the spontaneous release is the fluorescence from wells which only contain target cells and maximum release is the fluorescence from wells containing target cells and have been treated with 3% Lysol.
  • Cell cytotoxicity % specific lysis for the ARH-77 cell line is shown in Figure 24 .
  • the CD70+ expressing cell lines ARH-77, MEC-1, SU-DHL-6, IM-9 and HuT 78 showed antibody mediated cytotoxicity with the HuMAb anti-CD70 antibody 2H5 and an increased percentage of specific lysis associated with the defucosylated form of the anti-CD70 antibody 2H5.
  • anti-CD16 antibody was shown to block the ADCC effect in the MEC-1 cell line. This data demonstrates that defucosylated HuMAb anti-CD70 antibodies show increased specific cytotoxicity to CD70+ expressing cells.
  • Example 15 Assessment of ADCC activity of anti-CD70 antibody using a 51 Cr-release assay
  • an anti-CD70 monoclonal antibody was tested for the ability to kill CD70+ Raji B lymphocyte cells in the presence of effector cells via antibody dependent cellular cytotoxicity (ADCC) in a 51 Cr-release assay.
  • ADCC antibody dependent cellular cytotoxicity
  • Human peripheral blood mononuclear cells were purified from heparinized whole blood by standard Ficoll-paque separation. The cells were resuspended at 2x10 6 /mL in RPMI1640 media containing 10% FBS and 200 U/ml of human IL-2 and incubated overnight at 37°C. The following day, the cells were collected and washed once in culture media and resuspended at 2x10 7 cells/ml. Two million target Raji cells (human B lymphocyte Burkitt's lymphoma; ATCC Accession No. CCL-86) were incubated with 200 ⁇ Ci 51 Cr in 1 ml total volume for 1 hour at 37°C.
  • target Raji cells human B lymphocyte Burkitt's lymphoma; ATCC Accession No. CCL-86
  • the target cells were washed once, resuspended in 1ml of media, and incubated at 37°C for an additional 30 minutes. After the final incubation, the target cells were washed once and brought to a final volume of 1x10 5 cells/ml.
  • 100 ⁇ l of labeled Raji cells were incubated with 50 ⁇ l of effector cells and 50 ⁇ l of antibody.
  • a target to effector ratio of 1:100 was used throughout the experiments.
  • human IgG1 isotype control was used as a negative control.
  • the PBMC culture was separated equally into tubes containing either 20 ⁇ g/mL of an anti-human CD 16 antibody, an irrelevant mouse IgG1 antibody, or no antibody prior to adding PBMC to the assay plate. Following a 15 minute incubation at 27° C, the blood cells were used as described above without washing.Following a 4 hour incubation at 37° C, the supernatants were collected and counted on a Cobra II auto-gamma Counter (Packard Instruments) with a reading window of 240-400 keV. The counts per minute were plotted as a function of antibody concentration and the data was analyzed by non-linear regression, sigmoidal dose response (variable slope) using Prism software (San Diego, CA).
  • % Lysis (Sample CPM- no antibody CPM)/TritonX CPM-No antibody CPM) X 100.
  • An antibody titration curve for cell cytotoxicity % specific lysis for the Raji cell line is shown in Figure 25 .
  • This data demonstrates that anti-CD70 antibodies have an ADCC effect on the Raji cell line.
  • the EC 50 value for the anti-CD70 antibody against Raji cells was 36.61nM.
  • a graph of cytotoxicity on Raji cells in the presence of an anti-CD16 antibody is shown in Figure 26 . This data demonstrates that the ADCC effect of anti-CD70 antibodies on Raji cells is dependent upon CD16.
  • Example 16 Assessment of ADCC activity of anti-CD70 antibody on activated T cells
  • a defucosylated and non-defucosylated anti-CD70 monoclonal antibody was tested for the ability to kill activated T cells in the presence of effector cells via antibody dependent cellular cytotoxicity (ADCC) in a fluorescence cytotoxicity assay.
  • ADCC antibody dependent cellular cytotoxicity
  • Human Anti-CD70 monoclonal antibody 2H5 was defucosylated as described above.
  • Human effector cells were prepared as described above.
  • Human spleen T cells were positively selected with anti-CD3 coated magnetic beads (Purity >90%). The cells were stimulated with anti-CD3 and anti-CD28 coated beads and 25ng/ml IL-2 in Iscove's media + 10% heat inactivated FCS for 6 days. Cells were collected and assayed for viability by propidium iodide incorporation (60% viable) and live cells were gated and analyzed for CD70 expression ( ⁇ 65% CD70+ on live cells) prior to inclusion in ADCC assays.
  • the activated T cells were tested for antibody specific ADCC to the defucosylated and non-defucosylated human anti-CD70 monoclonal antibody 2H5 using the Delfia fluorescence emission analysis as follows.
  • the target activated T cells (100 ⁇ l of labeled target cells) was incubated with 50 ⁇ l of effector cells and 50 ⁇ l of either 2H5 or defucosylated 2H5 antibody.
  • a target to effector ratio of 1:50 was used throughout the experiments.
  • a human IgG1 isotype control was used as a negative control.
  • % lysis was calculated as follows: (sample release - spontaneous release * 100) / (maximum release - spontaneous release), where the spontaneous release is the fluorescence from wells which only contain target cells and maximum release is the fluorescence from wells containing target cells and have been treated with 3% Lysol. Cell cytotoxicity % specific lysis for the activated T cells is shown in Figure 27 .
  • the activated T cells showed antibody mediated cytotoxicity with the HuMAb anti-CD70 antibody 2H5 and an increased percentage of specific lysis associated with the defucosylated form of the anti-CD70 antibody 2H5.
  • the antibody mediated cytotoxicity was blocked by the addition of anti-CD 16 antibody in both the defucosylated and non-defucosylated forms of anti-CD70 antibody.
  • the control IgG had no effect on cytotoxicity. This data demonstrates that defucosylated HuMAb anti-CD70 antibodies show increased specific cytotoxicity to activated T cells.
  • Example 17 Blocking assay for receptor-ligand CD70-CD27 binding
  • anti-CD70 monoclonal antibodies were tested for their ability to block the interaction of CD70 with the ligand CD27 using a blocking assay.
  • Wells were coated overnight with 100 ⁇ l/well of an anti-IgG antibody (Fc-sp.) at 2 ⁇ g/ml at 4°C. The wells were blocked with 200 ⁇ l/well 1% BSA/PBS for 1 hour at room temperature. To each well was added 100 ⁇ l/well of CD27-Fc-his at 0.16 ⁇ g/ml for 1 hour at 37°C while shaking. Each well was washed 5 times with 200 ⁇ l/well PBS/Tween 20 (0.05 % (v:v)).
  • Fc-sp. an anti-IgG antibody
  • Anti-CD70 antibody was diluted in 10% NHS + 1% BSA/PBS and mixed with CD70-myc-his at 0.05 ⁇ g/ml, incubated for 1 hour at room temperature and washed 5 times with 200 ⁇ l/well PBS/Tween 20 (0.05 % (v:v)).
  • a known antibody that blocks CD70/CD27 interaction was used as a positive control and an isotype control antibody was used as a negative control.
  • the mixture of CD70 and anti-CD70 antibody was blocked with an anti-Fc antibody and 100 ⁇ l/well CD70-myc-his + antibody was added to the wells containing CD27-Fc-his. The mixture was incubated for 1 hour shaking at 37°C.
  • Example 18 Treatment of in vivo tumor xenograft model using naked anti-CD70 antibodies
  • mice implanted with a lymphoma tumor were treated in vivo with naked anti-CD70 antibodies to examine the in vivo effect of the antibodies on tumor growth.
  • ARH-77 human B lymphoblast leukemia; ATCC Accession No. CRL-1621
  • Raji human B lymphocyte Burkitt's lymphoma; ATCC Accession No. CCL-86
  • Male Ncr athymic nude mice (Taconic, Hudson, NY) between 6-8 weeks of age were implanted subcutaneously in the right flank with 5 x10 6 ARH-77 or Raji cells in 0.2 ml of PBS/Matrigel (1:1) per mouse. Mice were weighed and measured for tumors three dimensionally using an electronic caliper twice weekly after implantation. Tumor volumes were calculated as height x width x length/2.
  • mice with ARH-77 tumors averaging 80 mm 3 or Raji tumors averaging 170 mm 3 were randomized into treatment groups.
  • the mice were dosed intraperitoneally with PBS vehicle, isotype control antibody or naked anti-CD70 HuMAb 2H5 on Day 0. Mice were euthanized when the tumors reached tumor end point (2000 mm 3 ).
  • the results are shown in Figure 29A (Raji tumors) and 29B (ARH-77 tumors).
  • the naked anti-CD70 antibody 2H5 extended the mean time to reaching the tumor end point volume (2000 mm 3 ) and slowed tumor growth progression.
  • treatment with an anti-CD70 antibody alone has a direct in vivo inhibitory effect on tumor growth.
  • Example 19 Treatment of in vivo lymphoma tumor xenograft model using cytotoxin-conjugated anti-CD70 antibodies
  • mice implanted with a lymphoma tumor were treated in vivo with toxin-conjugated anti-CD70 antibodies to examine the in vivo effect of the antibodies on tumor growth.
  • ARH-77 human B lymphoblast leukemia; ATCC Accession No. CRL-1621
  • Granta 519 DSMZ Accession No. 342
  • Raji human B lymphocyte Burkitt's lymphoma; ATCC Accession No. CCL-86
  • Male Ncr athymic nude mice (Taconic, Hudson, NY) between 6-8 weeks of age were implanted subcutaneously in the right flank with 5 x10 6 ARH-77, 10 x10 6 Granta 519 or 5 x10 6 Raji cells in 0.2 ml of PBS/Matrigel (1:1) per mouse. Mice were weighed and measured for tumors three dimensionally using an electronic caliper twice weekly after implantation.
  • Tumor volumes were calculated as height x width x length/2. Mice with tumors averaging 80 mm 3 (ARH-77), 220 mm 3 (Granta 519), or 170 mm 3 (Raji), were randomized into treatment groups. The mice were dosed intraperitoneally with PBS vehicle, toxin-conjugated isotype control antibody or toxin-conjugated anti-CD70 HuMAb 2H5 on Day 0. Examples of toxin compounds that may be conjugated to the antibodies of the current disclosure are described in U.S. Provisional Application Serial No. 60/720,499 , filed on September 26, 2005. Mice were euthanized when the tumors reached tumor end point (2000 mm 3 ).
  • Example 20 Cross-reactivity of anti-CD70 antibody with rhesus B lymphoma cells
  • FACS analysis was also employed to access the ability of the anti-CD70 antibody 69A7 cross reacting with the monkey rhesus CD70+ B lymphoma cell line, LCL8664 (ATCC#: CRL-1805). Binding of the HuMAb 69A7 anti-CD70 human monoclonal antibody was assessed by incubating 1x10 5 cells with 69A7 at a concentration of 1 ⁇ g/ml. The cells were washed and binding was detected with a FITC-labeled anti-human IgG Ab. An isotype control antibody was used as a negative control. Flow cytometric analyses were performed using a FACSCalibur flow cytometry (Becton Dickinson, San Jose, CA). The results are shown in Figure 31 . The result demonstrated that the anti-CD70 antibody 69A7 cross-reacts with monkey CD70+ B lymphoma cells.
  • the 786-O human renal cancer cell line was used to test the internalization of HuMab anti-CD70 antibodies 69A7 and 2H5 upon binding to the cells using immuno-fluorescence staining.
  • 786-O cells (1x10 4 cells per 100 ⁇ l per well in a 96-well plate) were harvested from a tissue culture flask by treatment with 0.25% Trypsin/EDTA, then incubated with each of the HuMab anti-CD70 antibodies at 5 ⁇ g/ml in FACS buffer (PBS + 5% FBS, media) for 30 minutes on ice.
  • a human IgG1 isotype control was used as a negative control.
  • the cells were re-suspended in the media (100 ⁇ l per well) and then incubated with goat anti-human secondary antibody conjugated with PE (Jackson ImmunoResearch Lab) at 1:100 dilution on ice for 30 minutes.
  • the cells were either immediately imaged for morphology and immunofluorescence intensity under a fluorescent microscope (Nikon) at 0 min or incubated at 37° C for various times. Fluorescence was observed in the cells stained with HuMab anti-CD70 antibodies, but not in the control antibody. Similar results were also obtained with FITC-direct conjugated HuMab anti-CD70 antibodies in the assays.
  • Example 22 HuMAb anti-CD70 blocks the binding of a known mouse anti-CD70 antibody
  • the HuMAb anti-CD70 antibody 69A7 was tested for its ability to block binding of a known mouse anti-CD70 antibody to CD70+ renal carcinoma 786-O cells.
  • 786-O cells were incubated with the mouse anti-CD70 antibody BU-69 (Ancell, Bayport, MN) at 1 ⁇ g/ml and the HuMAb 69A7 at 1, 5 or 10 ⁇ g/ml for 20 minutes on ice.
  • IgG1 and IgG2 isotype control antibodies were used as negative controls. The cells were washed twice and binding was detected with a FITC-labeled anti-human IgG Ab.
  • Example 23 HuMAb anti-CD70 inhibits inflammatory response
  • CHO-S cells stably transfected with mouse CD32 (CHO-S/mCD32 cells) were transiently transfected with a full length human CD70 construct (CHO-S/mCD32/CD70 cells).
  • Surface expression was confirmed by flow cytometry using 2A5 and PE conjugated anti-human IgG secondary Ab (data not shown).
  • RosetteSep ® Human T Cell Enrichment Kit (Cat# 15061; StemCell Technologies Inc) purified human peripheral blood CD3+ T cells were stimulated in vitro at 1 x 10 6 /well with 1 x 10 5 CHO-S/mCD32 or CHO-S/mCD32/CD70 cells/well, 1 ⁇ g/ml anti-hCD3 (clone OKT3; BD Bioscience) and serial dilutions of either the HuMAb 2H5 or non-fucosylated 2H5 (2H5 NF) in triplicate wells of a 96 well plate.
  • 2H5 inhibition is specific to CD70 costimulation as 2H5 had no effect on anti-CD3 + CHO-S/mCD32 mediated proliferation. Both 2H5 and 2H5 NF completely inhibited CD70 co-stimulated INF- ⁇ secretion in a dose dependent manner as well ( Figure 33B ). Data also show 2H5 inhibition is specific to CD70 costimulation as 2H5 had no effect on anti-CD3 + CHO-S/mCD32 mediated INF- ⁇ secretion. Together data show 2H5 and 2H5 NF functionally block CD70 human T cell costimulation.
  • PBMC peripheral blood mononuclear cells
  • CMV cytomegalovirus
  • Astarte, Inc cytomegalovirus
  • Human MHC class I haplotype B*3501+ PBMC pre-screened for cytomegalovirus (CMV) specific T cell responses were cultured in the presence of 25 ng/ml of B*3501 binding CMV peptide IPSINVHHY (ProImmune) and 20 ⁇ gs/ml of the HuMAb 2H5 in the presence or absence of serial dilutions of an anti-human CD 16 (FcR ⁇ III) functional blocking Ab (clone 3G8; BD Biosciences) for 11 days and were then analyzed by flow cytometry for peptide specific CD8+ cell numbers as described above. The results are shown in Figure 35 .
  • CMV cytomegalovirus
  • Example 24 Treatment of in vivo renal carcinoma tumor xenograft model using cytotoxin-conjugated anti-CD70 antibodies
  • mice implanted with a renal carcinoma tumor were treated in vivo with toxin-conjugated anti-CD70 antibodies to examine the in vivo effect of the antibodies on tumor growth.
  • mice 786-O (ATCC Accession No. CRL-1932) and Caki-1 (ATCC Accession No. HTB-46) cells were expanded in vitro using standard laboratory procedures.
  • Male CB17.SCID mice (Taconic, Hudson, NY) between 6-8 weeks of age were implanted subcutaneously in the right flank with 2.5 million 786-0 or Caki-1 cells in 0.2 ml of PBS/Matrigel (1:1) per mouse. Mice were weighed and measured for tumors three dimensionally using an electronic caliper twice weekly after implantation. Tumor volumes were calculated as height x width x length. Mice with tumors averaging 200 mm 3 were randomized into treatment groups.
  • mice were dosed intraperitoneally with PBS vehicle, toxin-conjugated isotype control antibody or toxin-conjugated anti-CD70 HuMAb 2H5 on Day 0.
  • Examples of toxin compounds that may be conjugated to the antibodies of the current disclosure are described in U.S. Provisional Application Serial No. 60/720,499 , filed on September 26, 2005.
  • Mice were euthanized when the tumors reached tumor end point (2000 mm 3 ). The results are shown in Figure 36A (786-O), and 36B (Caki-1).
  • the anti-CD70 antibody 2H5 conjugated to a toxin extended the mean time to reaching the tumor end point volume (2000 mm 3 ) and slowed tumor growth progression.

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BRPI0617549A2 (pt) 2011-07-26
WO2007038637A3 (en) 2007-10-04
US20090028872A1 (en) 2009-01-29
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CA2623236A1 (en) 2007-04-05
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